Injectable nitrogen mustard compositions comprising a cyclodextrin derivative and methods of making and using the same

ABSTRACT

The present disclosure is directed to pharmaceutical compositions comprising a nitrogen mustard and a cyclodextrin derivative, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/229,523, filed Mar. 28, 2014, which is a continuation-in-part of U.S.patent application Ser. No. 14/108,169, filed Dec. 16, 2013, which is acontinuation of U.S. patent application Ser. No. 12/790,724, filed May28, 2010, which claims the benefit of the filing date of U.S. Appl. No.61/182,560, filed May 29, 2009, all of which are incorporated herein byreference in their entirety.

BACKGROUND Field

The present embodiments relate to pharmaceutical compositions comprisinga nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., and a cyclodextrinderivative, and methods of making and using the same, for example, totreat disorders and diseases that are therapeutically responsive to thenitrogen mustard.

BACKGROUND

Melphalan is an alkylating agent of the bischloroethylamine type, and isactive against both resting and rapidly dividing tumor cells. Aninjectable melphalan composition (ALKERAN® for Injection,GlaxoSmithKline) has been approved by the U.S. Food and DrugAdministration for the palliative treatment of patients with multiplemyeloma for whom oral therapy is not appropriate, and an oral melphalancomposition (ALKERAN® Tablets, GlaxoSmithKline) has been approved forthe palliative treatment of multiple myeloma and for the palliation ofnon-resectable epithelial carcinoma of the ovary.

ALKERAN® for Injection (GlaxoSmithKline) is administered intravenouslyafter first diluting a sterile, nonpyrogenic, freeze-dried powdercontaining melphalan hydrochloride (equivalent to 50 mg melphalan) and20 mg povidone with a sterile diluent that contains sodium citrate (0.2g), propylene glycol (6 mL), ethanol (96%, 0.52 mL), and water, for atotal volume of 10 mL. The usual intravenous dose is 16 mg/m², which isadministered as a single infusion over 15 to 20 minutes. Melphalan isintravenously administered in 4 doses at 2-week intervals, then, afteradequate recovery from toxicity, at 4-week intervals.

According to the ALKERAN® for Injection (GlaxoSmithKline) label,following administration of ALKERAN® for Injection, drug plasmaconcentrations of melphalan decline rapidly in a biexponential mannerwith distribution phase and terminal elimination phase half-lives ofapproximately 10 and 75 minutes, respectively. The average total bodyclearance is 7 to 9 mL/min/kg (250 to 325 mL/min/m²). A study hasreported that on repeat dosing of 0.5 mg/kg every 6 weeks, the clearanceof melphalan decreased from 8.1 mL/min/kg after the first course, to 5.5mL/min/kg after the third course, but did not decrease appreciably afterthe third course. Mean (±SD) peak melphalan plasma concentrations inmyeloma patients after administration of 10 or 20 mg/m² doses ofmelphalan were 1.2±0.4 and 2.8±1.9 μg/mL, respectively. Afterintravenous administration of 50 mg of melphalan, the steady-statevolume of distribution of melphalan is 0.5 L/kg. The extent of melphalanbinding to plasma proteins ranges from 60% to 90%. Serum albumin is themajor binding protein, while α₁-acid glycoprotein appears to account forabout 20% of the plasma protein binding. Approximately 30% of the drugis (covalently) irreversibly bound to plasma proteins. Interactions withimmunoglobulins have been found to be negligible.

Melphalan is eliminated from plasma primarily by chemical hydrolysis tomonohydroxymelphalan and dihydroxymelphalan. Aside from these hydrolysisproducts, no other melphalan metabolites have been observed in humans.

Controlled trials comparing intravenous to oral melphalan have showngreater myelosuppression with the intravenously administered melphalan.Furthermore, hypersensitivity reactions, including anaphylaxis, haveoccurred in approximately 2% of patients who have received intravenousmelphalan. Melphalan also undergoes rapid hydrolysis in aqueoussolution. Melphalan in the ALKERAN® For Injection (GlaxoSmithKline)product also rapidly forms a citrate derivative upon reconstitution andcannot be refrigerated due to precipitation of melphalan from solution.

Melphalan compositions comprising a cyclodextrin derivative as a carrierand/or a diluent are known.

BRIEF SUMMARY

What may be useful is a melphalan formulation that can minimize thetoxicology and side-effect profile of intravenous melphalan. What mayalso be useful is an intravenous melphalan formulation having increasedbioavailability and/or an improved rate of therapeutic onset. Alsopotentially useful is a melphalan composition suitable for intravenousadministration that is stable under ambient and/or refrigeratedconditions, and can provide fully dissolved melphalan without the needfor organic solubilizers (e.g., ethanol and/or propylene glycol, and thelike). What may also be helpful is a composition free from componentsthat rapidly form a derivative with melphalan. What may also be helpfulis a melphalan composition suitable for intravenous administration thathas improved stability, thereby enabling longer duration infusions, andlengthening the melphalan exposure time that a patient receives from asingle, convenient administration. As described herein, compositionssuitable for oral or parenteral administration that include melphalanand a cyclodextrin derivative have been developed.

Some embodiments include a pharmaceutical composition comprising 25 mgto 125 mg of a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., as a hydrochloridesalt, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6. In someembodiments, the nitrogen mustard comprises melphalan wherein dilutionof the pharmaceutical composition with an aqueous solution provides amelphalan solution ready for infusion in which the melphalan degrades by2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C.within 10 hours after the dilution; and wherein the cyclodextrinderivative is present in a ratio of 50:1 to 100:1 (w/w) relative to themelphalan.

Some embodiments include pharmaceutical composition comprising 150 mg,to 250 mg of a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., as a hydrochloridesalt, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6. In someembodiments, the nitrogen mustard comprises melphalan, wherein dilutionof the pharmaceutical composition with an aqueous solution provides amelphalan solution ready for infusion in which the melphalan degrades by2% or less at about 25° C. within 5 hours, or 4% or less at about 25° C.within 10 hours after the dilution, and wherein the cyclodextrinderivative is present in a ratio of 25:1 to 35:1 (w/w) relative to themelphalan.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄ ⁻SO₃ ⁻Na⁺, and x=6.0-7.1.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1; and

-   -   the pharmaceutical composition comprises about 50 mg of        melphalan as a hydrochloride salt and the cyclodextrin        derivative is present in a concentration of 50:1 to 100:1 (w/w)        relative to the melphalan; or    -   the pharmaceutical composition comprises about 50 mg of        melphalan as a hydrochloride salt the cyclodextrin derivative is        present in a ratio of about 55:1 (w/w) relative to the        melphalan; or    -   the pharmaceutical composition comprises about 200 mg of        melphalan as a hydrochloride salt and the cyclodextrin        derivative is present in a ratio of 25:1 to 35:1 (w/w) relative        to the melphalan; or    -   the pharmaceutical composition comprises about 200 mg of        melphalan as a hydrochloride salt and the cyclodextrin        derivative is present in a ratio of about 27:1, about 30:1, or        about 32:1 (w/w) relative to the melphalan.

The present disclosure is directed to a method of treating a subjectsuffering from a neoplastic disorder, the method comprising diluting acomposition with an aqueous diluent to provide a dilute pharmaceuticalcomposition comprising 25 mg to 125 mg of a nitrogen mustard, such asmelphalan, mechlorethamine, cyclophosphamide, ifosfamide, bendamustine,etc., and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thedilute pharmaceutical composition has a pH of about 4 to about 6. Insome embodiments, the nitrogen mustard comprises melphalan, wherein thecyclodextrin derivative is present in a concentration of at, least 50:1(w/w) relative to the melphalan; wherein the melphalan in the dilutepharmaceutical composition degrades by 2% or less at about 25° C. within5 hours, or 4% or less at about 25° C. within 10 hours after thediluting; and administering the dilute pharmaceutical composition byinjection to the subject in need thereof.

In some embodiments, the neoplastic disorder is: myeloma, multiplemyeloma, acute myelogenous leukemia, melanoma, malignant melanoma,breast cancer, ovarian cancer, testicular cancer, advanced prostatecancer, a neuroendocrine cancer, metastatic melanoma (e.g., metastaticocular melanoma, metastatic cutaneous melanoma, and the like), ametastatic neuroendocrine tumor, a metastatic adenocarcinoma tumor,hepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma,plasma cell neoplasm, amyloidosis, scleromyxedema, or a combinationthereof. In some embodiments, the neoplastic disorder is multiplemyeloma and the administering is systemic and provides palliativetreatment of the multiple myeloma.

Some embodiments include a method for conditioning a subject in need ofa stem cell transplantation, the method comprising administering anitrogen mustard, such as melphalan, at a dose of 50 mg/m² to 300 mg/m²per day to the subject in need of the stem cell transplantation, whereinthe nitrogen mustard is administered in a pharmaceutical compositioncomprising the nitrogen mustard and a cyclodextrin derivative of formulaI:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has, a pH of about 4 to about 6. In someembodiments, the nitrogen mustard is melphalan and the cyclodextrinderivative is present in a ratio of at least 25:1 (w/w) relative to themelphalan.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ is a hydroxy-substituted-C₃ group.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ areindependently a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ grouphaving a degree of substitution of 4 to 8 per cyclodextrin derivative,and the remaining substituents are —H.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ is substituted with a straight-chain C₄-(alkylene)-SO₃ ⁻ group.

In some embodiments, a pharmaceutical composition or a dilutepharmaceutical composition is substantially free of an alcohol.

In some embodiments, the aqueous diluent is a saline solution.

In some embodiments, a dilute pharmaceutical composition is stored about0.5 hours to about 48 hours prior to the administering. In someembodiments, the nitrogen mustard, such as melphalan, in apharmaceutical composition degrades by 2% or less at about 25° C. within5 hours, or 4% or less at about 25° C. within 10 hours after thediluting.

In some embodiments, a subject suffering from a neoplastic disorder orin need of a stem cell transplantation is a pediatric subject.

In some embodiments, the administering is performed intravenously. Insome embodiments, the administering is performed via a limb perfusion.

In some embodiments, the administering is for a period of two or moredays.

In some embodiments, the administering provides a nitrogen mustardC_(max) in a subject that is at least 20% or greater than a nitrogenmustard C_(max) provided by a nitrogen mustard formulation containing anequivalent dose of nitrogen mustard and lacking the cyclodextrinderivative. In some embodiments, the administering provides a nitrogenmustard AUC_(0-t) in a subject that is at least 20% or greater than anitrogen mustard AUC_(0-t) provided by a nitrogen mustard formulationcontaining an equivalent dose of nitrogen mustard and lacking thecyclodextrin derivative.

In some embodiments, the administering provides a melphalan C_(max) in asubject that is at least 20% or greater than a melphalan C_(max)provided by a melphalan formulation containing an equivalent dose ofmelphalan and lacking the cyclodextrin derivative. In some embodiments,the administering provides a melphalan AUC_(0-t) in a subject that is atleast 20% or greater than a melphalan AUC_(0-t) provided by a melphalanformulation containing an equivalent dose of melphalan and lacking thecyclodextrin derivative.

Some methods comprise diluting a concentrated nitrogen mustardcomposition, such as a composition of melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., with an aqueousdiluent to provide the pharmaceutical composition. In some embodiments,a concentrated nitrogen mustard composition comprises 50 mg to 500 mg ofthe nitrogen mustard. In some embodiments, a concentrated melphalancomposition comprises about 200 mg of the nitrogen mustard.

Some methods comprise diluting a concentrated melphalan composition withan aqueous diluent to provide the pharmaceutical composition. In someembodiments, a concentrated melphalan composition comprises 50 mg to 500mg of melphalan. In some embodiments, a concentrated melphalancomposition comprises about 200 mg of melphalan.

In some embodiments, a first container comprises povidone in an amountof 10 mg to 30 mg, and a second container comprises a pH-adjusting agentin a concentration sufficient to provide a pH of about 4 to about 6 whenthe first container and the second container are combined.

In some embodiments, the cyclodextrin derivative present in the secondcontainer is a compound of formula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1; wherein the first container comprises about200 mg of a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., as a hydrochloridesalt; and wherein the cyclodextrin derivative is present in the secondcontainer in an amount of about 27:1, about 30:1, or about 32:1 (w/w)relative to the nitrogen mustard.

Some embodiments include a pharmaceutical kit comprising a firstcontainer comprising 25 mg to 125 mg of melphalan as a hydrochloridesalt and an optional water-soluble polymer, and a second containercomprising an aqueous diluent, an optional buffer, and a cyclodextrinderivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of at least 50:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours, or 4% orless at about 25° C. within 10 hours after the diluting.

Some embodiments include a pharmaceutical kit comprising a firstcontainer comprising 150 mg to 250 mg of melphalan as a hydrochloridesalt and an optional water-soluble polymer; and a second containercomprising an aqueous diluent, an optional buffer, and a cyclodextrinderivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of 25:1 to 35:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours, or 4% orless at about 25° C. within 10 hours after the diluting.

In some embodiments, a first container comprises povidone in an amountof 10 mg to 30 mg, and a second container comprises a pH-adjusting agentin a concentration sufficient to provide a pH of about 4 to about 6 whenthe first container and the second container are combined.

In some embodiments, the cyclodextrin derivative present in the secondcontainer is a compound of formula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1; wherein the first container comprises about200 mg of melphalan as a hydrochloride salt; and wherein thecyclodextrin derivative is present in the second container in an amountof about 27:1, about 30:1, or about 32:1 (w/w) relative to themelphalan.

Further embodiments, features, and advantages of the presentembodiments, as well as the composition, structure and operation of thevarious embodiments, are described in detail below with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate one or more embodiments and, togetherwith the description, further serve to explain the principles of theembodiments and to enable a person skilled in the pertinent art to makeand use various embodiments.

FIG. 1 provides a graphic representation of the solubility of free basemelphalan as a function of pH and the concentration of a cyclodextrinderivative.

FIG. 2 provides a graphic representation of the solubility of free basemelphalan and melphalan hydrochloride at pH 7.5 as a function of theconcentration of a cyclodextrin derivative.

FIGS. 3 and 4 provide flow charts that describe processes for preparinga unit dosage form.

FIGS. 5A-5B provide a graphic representation of dose-normalized wholeblood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalanfollowing intravenous administration to male Sprague-Dawley rats using amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and a cyclodextrin-free melphalan formulation (ALKERAN®for Injection, GlaxoSmithKline).

FIG. 6 provides a graphic representation of the mean plasma melphalanconcentration in a human patient after intravenous administration of amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and after intravenous administration of acyclodextrin-free melphalan formulation (Melphalan HCl Injectable,Bioniche Pharma USA).

One or more embodiments of the present invention will now be describedwith reference to the accompanying drawings. In the drawings, likereference numbers can indicate identical or functionally similarelements. Additionally, the left-most digit(s) of a reference number canidentify the drawing in which the reference number first appears.

DETAILED DESCRIPTION

Throughout the specification, use of the term “about” with respect toany quantity is contemplated to include that quantity. For example,“about 10 mL” is contemplated herein to include “10 mL,” as well asvalues understood in the art to be approximately 10 mL with respect tothe entity described.

Combinations and sub-combinations of the various aspects and embodimentsdisclosed herein are specifically contemplated. Further, when aparticular feature, structure, or characteristic is described inconnection with an embodiment, it is understood that it is within theknowledge of one skilled in the art to effect such feature, structure,or characteristic in connection with other embodiments whether or notexplicitly described. These and other embodiments will be apparent uponreference to the following detailed description, examples, claims andattached figures.

Nitrogen Mustards

A composition, formulation, or unit dosage form can include a nitrogenmustard compound, such as mechlorethamine, mustine, cyclophosphamide,chlorambucil, uramustine, ifosfamide, melphalan, bendamustine, etc. Anitrogen mustard having one or more chiral centers could include anenantiomerically or diastereomerically pure form any isomer, or couldcontain any mixture of isomers. A nitrogen mustard may also be in analternate solid form, such as an addition salt, a polymorph, a solvate,a hydrate, a dehydrate, a co-crystal, an anhydrous form, and or anamorphous form, etc.

Melphalan

In some embodiments, a composition, formulation or unit dosage form cancomprise melphalan, which has the following chemical structure:

As used herein, the term “melphalan” refers to the L-isomer of the abovecompound, 4-[bis(chloroethyl)amino]phenylalanine, as well as additionsalts, polymorphs, solvates, hydrates, dehydrates, co-crystals,anhydrous, and amorphous forms thereof. Melphalan contains a chiralatom, and thus, as used herein, “melphalan” can refer to thesubstantially pure form of the L-isomer. As used herein, “substantiallypure” refers to melphalan having a purity of 90% or higher, 95% orhigher, 98% or higher, 99% or higher, 99.5% or higher, or 99.9% orhigher.

The D-isomer of the above compound, known as melphalan, is less activeagainst certain animal tumors, and the dose needed to produce effects onchromosomes is larger than that required with melphalan. The racemic(DL-) form is known as merphalan or sarcolysin. Some melphalancompositions are substantially free of merphalan. In some embodiments,melphalan is present as a hydrochloride salt having a purity of 95% orgreater, 98% or greater, 99% or greater, 99.9% or greater, or 99.99% orgreater.

Melphalan is a bifunctional alkylating agent that is active againstselected human neoplastic diseases. The molecular formula for melphalanis C₁₃H₁₈Cl₂N₂O₂, and the molecular weight of the free base form is305.20 g/mol. Melphalan is practically insoluble in water (pH 7) and hasa pK_(a) of about 2.5.

In some embodiments, the pharmaceutical compositions and dosage formscomprise a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc. as a hydrochloridesalt. As used herein, reference to a nitrogen mustard as a hydrochloridesalt, e.g. “melphalan as a hydrochloride salt,” refers to thehydrochloric acid addition salt of the nitrogen mustard compound.However, amounts and concentrations of a nitrogen mustard are providedin reference to an equivalent mass of free base of the nitrogen mustard.For example, 5 mg of “melphalan as a hydrochloride salt” refers to 5 mgof the active agent melphalan, exclusive of the hydrochloride additionsalt, which if considered would provide a total mass of about 5.6 mg.Reference to an amount of a nitrogen mustard refers to the equivalentamount of the free base, but does not limit the form of the nitrogenmustard. For example, “5 mg of melphalan” could include 5 mg ofmelphalan free base, 5.6 mg of the hydrochloride addition salt ofmelphalan, or an equivalent amount of another salt of melphalan.

With respect to any pharmaceutical product or composition comprising anitrogen mustard, such as melphalan, mechlorethamine, cyclophosphamide,ifosfamide, bendamustine, etc. and a cyclodextrin derivative, in someembodiments a single vial of the product or composition contains about10 mg to about 500 mg, about 50 mg to about 200 mg, about 50 mg to about100 mg, about 50 mg, about 100 mg, or about 150 mg of a nitrogenmustard, such as melphalan, mechlorethamine, cyclophosphamide,ifosfamide, bendamustine, etc.

Cyclodextrin Derivatives

The compositions, formulations and/or unit dosage forms comprise acyclodextrin derivative, such as a water-soluble cyclodextrinderivative. As used herein, “cyclodextrin derivative” includes a cyclicoligosaccharide comprising five or more α-D-glucopyranoside units linkedin a circular 1→4 configuration, and comprising a substituent groupattached to one or more of the glucopyranoside units at the 2, 3 and/or6 position(s) through an ether bond (—O—R—, where R refers to thesubstituent group).

In some embodiments, the cyclodextrin derivative is a compound offormula I:

wherein n is 4, 5 or 6, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently selected from: —H, a straight-chain or branchedC₁-C₈-(alkylene)-SO₃ ⁻ group, and an optionally substitutedstraight-chain or branched C₁-C₆ group. Compounds of Formula 1 can oftenbe water soluble.

In some embodiments, a cyclodextrin is selected based upon an averagedegree of substitution (“ADS”), which as used herein refers to theaverage number of substituent groups per cyclodextrin molecule. Theaverage degree of substitution for cyclodextrin derivatives is describedin detail in WO 2009/018069, which is incorporated herein by referencein its entirety. As used herein, a cyclodextrin derivative compositionis referred to by the following notation: the substituent(s) areabbreviated (e.g., sulfobutyl ether groups are abbreviated as “SBE”)with a subscript denoting the ADS of the substituent, and cyclodextrinstructure is defined. For example, a sulfobutyl ether-derivatizedβ-cyclodextrin composition having an ADS of 6.5 is referred to as“SBE_(6.5)-β-CD.” As a second example, a (β-cyclodextrin compositioncomprising cyclodextrin molecules derivatized with both sulfobutyl etherand hydroxypropyl groups is referred to as “SBE_(4.2)-HP_(2.5)-β-CD,”wherein the ADS of the sulfobutyl ether groups is 4.2 and the ADS of thehydroxypropyl groups is 2.5.

Cyclodextrin derivatives suitable for use include cyclodextrincompositions bearing substituent groups (R₁-R₉ and R in formulas I andII, respectively) that are independently selected from: —H, astraight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group, and anoptionally substituted straight-chain or branched C₁-C₆ group.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ of formula I is substituted with a straight-chain C₄-(alkylene)-SO₃ ⁻group. Exemplary C₁-C₈-(alkylene)-SO₃ ⁻ groups suitable for use include,but are not limited to, sulfoethyl, sulfopropyl, 1-methyl-sulfopropyl,sulfobutyl, 1-methyl-sulfobutyl, 2-methyl-sulfobutyl,1-methyl-sulfobut-3-yl, 2-ethyl-sulfobutyl, 3-ethyl-sulfobutyl,sulfopentyl, 1-sulfopent-3-yl, sulfohexyl, sulfoheptyl, sulfooctyl, andthe like, and combinations thereof.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ of formula Iare independently a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻group having an ADS of 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.5, 4.5 to 8, 4.5to 7.5, 4.5 to 7, 5 to 8, 5 to 7.5, 5 to 7, 5.5 to 8, 5.5 to 7.5, 5.5 to7, 5.5 to 6.5, 6 to 8, 6 to 7.5, 6 to 7.1, 6.5 to 7, about 6.5, or about7 per cyclodextrin derivative, and the remaining substituents are —H.

In some embodiments, a substituent is an optionally substitutedstraight-chain or branched C₁-C₆ group. As used herein, “optionallysubstituted” refers to one or more optional substituents such as:halogen (i.e., —F, —Cl, —Br, —I), —NO₂, —C≡N, —OR₂₂, —SR₂₂, —SO₂R₂₂,—C(═O)OR₂₂, —C(═O)R₂₂, —C(═O)N(R₂₂)₂, —SO₂N(R₂₂)₂, —SO₂N(H)C(═O)R₂₂,—SO₂N(H)C(═O)OR₂₂ (wherein R₂₂ is not H), —N(R₂₂)₂, —N(R₂₂)SO₂R₂₂,—N(R₂₂)C(O)_(m)R₂₂ (wherein m=1 or 2), —N(R₂₂)C(O)N(R₂₂)₂,—N(R₂₂)SO₂N(R₂₂)₂, —O—C(═O)R₂₂, —O—C(═O)OR₂₂, —O—C(═O)N(R₂₂)₂,—C(═O)N(H)SO₂N(R₂₂)₂, —C(═O)N(H)SO₂R₂₂, oxo (or keto, i.e., ═O), thioxo(i.e., ═S), imino (i.e., ═NR₂₂), —NR₂₂—C(═NR₂₂)R₂₂,—NR₂₂—C(═NR₂₂)N(R₂₂)₂, —C(═NR₂₂)N(R₂₂)₂, —O—C(═NR₂₂)N(R₂₂)₂,—O—C(═NR₂₂)R₂₂, —C(═NR₂₂)R₂₂, —C(═NR₂₂)OR₂₂, and ionic forms thereof(e.g., —N⁺(R₂₂)₂X⁻, and the like, wherein X⁻ is a pharmaceuticallyacceptable anion), wherein R₂₂ is independently selected at eachoccurrence from H, and C₁-C₄ alkyl.

Exemplary optionally substituted straight-chain or branched C₁-C₆ groupsinclude, but are not limited to, 2-hydroxypropyl, 3-hydroxypropyl,2,3-dihydroxypropyl, 3-oxobutyl, and 2-ethoxy-ethyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ of formula I is a hydroxy-substituted-C₃ group. In some embodiments,the cyclodextrin derivative comprises β-cyclodextrin that includes ahydroxy-substituted-C₃ group having an ADS of 1 to 8, 2 to 8, 3 to 7, 4to 7.5, 4.3 to 7.5, about 1, about 2, about 2.5, about 3, about 3.5,about 4, about 4.3, about 5, about 5.5, about 6, about 6.5, about 7, orabout 7.5.

Exemplary cyclodextrin compositions, and methods of making the same,also include those described in U.S. Pat. Nos. 5,134,127, 5,241,059,5,376,645, 5,874,418, 6,046,177, 6,133,248, 6,153,746, 6,204,256,7,034,013, 7,629,331, and 7,635,773, U.S. Pub. No. 2009/0012042, and PCTPub. No. WO 2005/117911, the contents of each of which is incorporatedherein by reference in the entirety.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺. In some embodiments, x=6.0-7.1. In some embodiments, thecyclodextrin derivative of formula II has an average molecular weight ofabout 2163 g/mol.

In some embodiments, the water soluble cyclodextrin derivative is ansulfobutylether-β-cyclodextrin [(SBE)-β-cyclodextrin]. In someembodiments, an (SBE)-β-cyclodextrin can have an ADS of about 1 to about10, about 3 to about 10, about 5 to about 9, about 6 to about 8, about 6to about 7, or about 6.5. For convenience, with respect to anypharmaceutical product or composition comprising a nitrogen mustardcompound, such as melphalan, and a cyclodextrin derivative, any suitablecyclodextrin derivative can be referred to as an“(SBE)_(x m)-β-cyclodextrin, wherein x refers to the ADS. For example,(SBE)_(6.5 m)-β-cyclodextrin has an ADS of about 6.5.

In some embodiments, the cyclodextrin derivative is a sulfobutylether-β-cyclodextrin having an ADS of about 7 (e.g., CAPTISOL®, CyDexPharmaceuticals, Inc., Lenexa, Kans.). CAPTISOL® cyclodextrin is apolyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cyclodextrin cavity by a butyl etherspacer group, or sulfobutylether (SBE). CAPTISOL® cyclodextrin has beenshown to be safe when administered parenterally, orally, or viainhalation and does not exhibit the nephrotoxicity associated with(β-cyclodextrin. Relative to β-cyclodextrin, CAPTISOL® sulfoalkyl ethercyclodextrin provides comparable or higher complexation characteristicsand superior water solubility in excess of 90 g per 100 mL, a 50-foldimprovement. Melphalan has a low binding affinity with CAPTISOL®(K_(a)=3×10² M⁻¹).

In some embodiments, the cyclodextrin derivative includes a substituentthat bears an ionic group that can optionally form a salt with apharmaceutically acceptable anion or cation. Pharmaceutically acceptablecations suitable for forming salts with negatively charged cyclodextrinderivatives include, but are not limited to, H⁺, Li⁺, Na⁺, K⁺, Mg²⁺,Ca²⁺, ammonium and amine cations such as cations of (C₁-C₆)-alkylamines,(C₄-C₈)-cycloalkylamines (e.g., piperidine, pyrazine, and the like),(C₁-C₆)-alkanolamines, and (C₄-C₈)-cycloalkanolamines, and the like, andcombinations thereof. In some embodiments, a pharmaceutically acceptablecation is Na⁺. Pharmaceutically acceptable anions suitable for formingsalts with positively charged cyclodextrin derivatives include, but arenot limited to, halides (e.g., Cl⁻ and the like), anions of(C₁-C₆)-alkyl acids (e.g., acetate, oxalate, fumarate, succinate, andthe like, and combinations thereof.

In some embodiments, the water soluble cyclodextrin derivative cancomprise a combination of two different types of cyclodextrinderivatives, such as sulfoalkylether β-cyclodextrin (i.e.sulfobutylether β-cyclodextrin) and hydroxyalkyl β-cyclodextrin (i.e.hydroxypropyl β-cyclodextrin). In some embodiments, the water solublecyclodextrin derivative is a mixture of sulfobutylether β-cyclodextrinand hydroxypropyl β-cyclodextrin. The two types of cyclodextrin ratiomay have a molar ratio of about 4 (4 moles sulfoalkyletherβ-cyclodextrin to 1 mole hydroxyalkyl β-cyclodextrin) to about 0.25,about 2 to about 0.5, about 1.2 to about 0.8, or about 1. In someembodiments, such a mixture may have a cyclodextrin to nitrogen mustardratio (e.g. cyclodextrin to melphalan ratio) of about 5 (5 molescyclodextrin to 1 mole nitrogen mustard such as melphalan) to about 100,about 7 to about 20, about 8 to about 12, or about 10.

With respect to any pharmaceutical product or composition comprising anitrogen mustard, such as melphalan, mechlorethamine, cyclophosphamide,ifosfamide, bendamustine, etc., and a water-soluble cyclodextrinderivative, any suitable amount of water-soluble cyclodextrin derivativecan be used. In some embodiments, a single vial of pharmaceuticalproduct or composition can contain about 1000 mg to about 5000 mg, about2000 mg to about 4000 mg, about 2000 mg to about 3000 mg, or about 2500mg to about 3000 mg of the water-soluble cyclodextrin derivative.

With respect to any pharmaceutical product or composition comprising anitrogen mustard, such as melphalan, mechlorethamine, cyclophosphamide,ifosfamide, bendamustine, etc., and a cyclodextrin derivative, anysuitable ratio of nitrogen mustard compound and cyclodextrin derivativemay be used. In some embodiments, a cyclodextrin derivative, such as awater-soluble cyclodextrin derivative [e.g. an (SBE)-β-cyclodextrin],and a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., can have a weightratio (cyclodextrin derivative:nitrogen mustard) of at least about 50:1,about 50:1 to about 100:1, about 55:1 to about 60:1, about 50:1, about55:1, or about 60:1.

With respect to any pharmaceutical product or composition comprising anitrogen mustard and a water-soluble cyclodextrin derivative, in someembodiments, a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., and the water-solublecyclodextrin derivative are present in a solid. With respect to anycomposition comprising a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc., and acyclodextrin derivative, the composition may have any suitable pH, suchas about 2 to about 8, about 4 to about 6, about 5 to about 6, or about5 to about 5.5.

Pharmaceutical Compositions and Unit Dosage Forms

The present disclosure is directed to pharmaceutical compositions andunit dosage forms comprising a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc., and acyclodextrin derivative, e.g. the nitrogen mustard and the cyclodextrincan be present together in a single vial. The pharmaceuticalcompositions of the present disclosure are suitable for parenteraladministration to a subject. Parenteral administration of thepharmaceutical compositions can include, but is not limited to, aninjection. Because parenteral administration can bypass a subject'snatural defenses against contaminants, the pharmaceutical compositionsare sterile or capable of being sterilized prior to administration.

Exemplary pharmaceutical compositions include, but are not limited to,solutions, suspensions or emulsions ready for administration, solutions,suspensions or emulsions ready to be dissolved in and/or diluted with apharmaceutically acceptable vehicle, and dry products ready to bedissolved in and/or diluted with a pharmaceutically acceptable vehicle.

Generally, the pharmaceutical compositions of the present disclosurecomprise a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., in a concentrationsuitable for treating a condition that is amenable to treatment with thenitrogen mustard. Thus, the pharmaceutical compositions of the presentdisclosure can be used to prepare a unit dosage form comprising atherapeutically effective amount of a nitrogen mustard for administeringto a subject in need thereof. Some embodiments include a unit dosageform that comprises a nitrogen mustard in a concentration that issuitable for administration without dilution. Alternatively, a unitdosage form of the present disclosure can be diluted prior toadministration to a subject in need thereof.

The present disclosure is also directed to a pharmaceutical compositioncomprising 25 mg to 125 mg, 25 mg to 100 mg, 25 mg to 75 mg, 25 mg to 50mg, 50 mg to 125 mg, 50 mg to 100 mg, 75 to 125 mg, 100 to 125 mg, about25 mg, about 50 mg, about 75 mg, about 100 mg, or about 125 mg of anitrogen mustard, such as melphalan, as a hydrochloride salt, anoptional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and K₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, about 4 toabout 5, about 4.5 to about 6, about 5 to about 6, about 5.5 to about 6,about 4, about 4.5, about 5, about 5.5, or about 6. In some embodiments,the nitrogen mustard comprises melphalan, wherein dilution of thepharmaceutical composition with an aqueous solution provides a solutionin which the melphalan degrades by 2% or less at about 25° C. within 5hours, or by 4% or less at about 25° C. within 10 hours after thedilution; and wherein the cyclodextrin derivative is present in a ratioof 50:1 to 100:1, 55:1 to 60:1, about 50:1, about 55:1, or about 60:1(w/w) relative to the melphalan.

The present disclosure is also directed to a pharmaceutical compositioncomprising 150 mg to 300 mg, 150 mg to 250 mg, 150 mg to 225 mg, 175 mgto 250 mg, 200 mg to 250 mg, about 150 mg, about 175 mg, about 200 mg,about 225 mg, or about 250 mg of a nitrogen mustard, such as melphalan,as a hydrochloride salt, an optional buffer, and a cyclodextrinderivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6. In someembodiments, the nitrogen mustard comprises melphalan wherein dilutionof the pharmaceutical composition with an aqueous solution provides amelphalan solution in which the melphalan degrades by 2% or less atabout 25° C. within 5 hours, or 4% or less at about 25° C. within 10hours after the dilution; and wherein the cyclodextrin derivative ispresent in a ratio of 25:1 to 35:1, about 27:1, about 30:1, or about32:1 (w/w) relative to the melphalan.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1; and

the pharmaceutical composition comprises about 50 mg of a nitrogenmustard, such as melphalan, as a hydrochloride salt and the cyclodextrinderivative is present in a concentration of 50:1 to 100:1, 55:1 to 60:1,about 50:1, about 55:1, or about 60:1 (w/w) relative to the nitrogenmustard; or

the pharmaceutical composition comprises about 200 mg of the nitrogenmustard, such as melphalan, as a hydrochloride salt and the cyclodextrinderivative is present in a ratio of 25:1 to 35:1, about 27:1, about30:1, or about 32:1 (w/w) relative to the nitrogen mustard.

Sterile solutions, suspensions, emulsions and the like can be preparedby incorporating a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., into an appropriatesolvent or carrier with the other optional ingredients enumeratedherein, followed by sterilization. Sterile powders can be prepared byspray drying, aseptic spray drying, vacuum drying, or freeze drying asterile solution, suspension, or emulsion to provide a dried solid(e.g., a powder) comprising a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc., alongwith any additional excipients.

Some embodiments include a solid pharmaceutical composition consistingof about 50 mg of a nitrogen mustard, such as melphalan, as ahydrochloride salt, an amount sufficient of an acid, a base, or acombination thereof to provide a pH of about 4 to about 6 upon dilutionwith a saline solution to a volume of about 10 mL, and a cyclodextrinderivative of formula II

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x-6.0-7.1, wherein dilution of the solid pharmaceuticalcomposition with an aqueous solution provides a nitrogen mustardsolution in which the nitrogen mustard has improved stability, andwherein the cyclodextrin derivative is present in a ratio of about 55:1(w/w) relative to the nitrogen mustard.

In some embodiments, the molar ratio of water soluble cyclodextrinderivative to nitrogen mustard, such as melphalan, is about 7 (10 molescyclodextrin to 1 mole nitrogen mustard) to about 100, about 10 to about20, about 9 to about 11, or about 10.

Some embodiments include a solid pharmaceutical composition consistingof about 50 mg of melphalan as a hydrochloride salt, an amountsufficient of an acid, a base, or a combination thereof to provide a pHof about 4 to about 6 upon dilution with a saline solution to a volumeof about 10 mL, and a cyclodextrin derivative of formula II

wherein 21-x of the R groups are H and x of the R groups are −(CH₂)₄⁻SO₃ ⁻Na⁺, and x-6.0-7.1, wherein dilution of the solid pharmaceuticalcomposition with an aqueous solution provides a melphalan solution inwhich the melphalan degrades by 2% or less at about 25° C. within 5hours, or by 4% or less at about 25° C. within 10 hours after thediluting, and wherein the cyclodextrin derivative is present in a ratioof about 55:1 (w/w) relative to the melphalan.

In some embodiments, the present disclosure is directed to a solidpharmaceutical composition consisting of about 200 mg of a nitrogenmustard, such as melphalan, as a hydrochloride salt, an amountsufficient of an acid, a base, or a combination thereof to provide a pHof about 4 to about 6 upon dilution with a saline solution to a volumeof about 20 mL, and a cyclodextrin derivative of formula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1, wherein dilution of the solid pharmaceuticalcomposition with an aqueous solution provides a nitrogen mustardsolution in which the nitrogen mustard has improved stability, andwherein the cyclodextrin derivative is present in a ratio of about 27:1,about 30:1, about 32:1, or about 50:1 (w/w), or greater, relative to thenitrogen mustard.

In some embodiments, the present disclosure is directed to a solidpharmaceutical composition consisting of about 200 mg of melphalan as ahydrochloride salt, an amount sufficient of an acid, a base, or acombination thereof to provide a pH of about 4 to about 6 upon dilutionwith a saline solution to a volume of about 20 mL, and a cyclodextrinderivative of formula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃Na⁺, and x=6.0-7.1, wherein dilution of the solid pharmaceuticalcomposition with an aqueous solution provides a melphalan solution inwhich the melphalan degrades by 2% or less at about 25° C. within 5hours, or by 4% or less at about 25° C. within 10 hours after thediluting, and wherein the cyclodextrin derivative is present in a ratioof about 27:1, about 30:1, about 32:1, or about 50:1 (w/w), or greater,relative to the melphalan.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a solid (e.g., a powder) or a liquid solution that is dilutedwith a liquid carrier or diluent prior to administration to a subject.Thus, the pharmaceutical compositions and unit dosage forms includesterile aqueous solutions, suspensions and dispersions, as well assterile solids (e.g., powders) comprising a nitrogen mustard, such asmelphalan, mechlorethamine, cyclophosphamide, ifosfamide, bendamustine,etc., that can be extemporaneously diluted or solubilized to provide asterile solution, suspension or dispersion.

In some embodiments, the compositions, formulations and/or unit dosageforms comprise a pharmaceutically acceptable excipient. As used herein,“pharmaceutically acceptable” refers to those excipients, compounds,materials, and/or compositions which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other possible complications commensurate with a reasonablebenefit/risk ratio.

In some embodiments, the pharmaceutical compositions and unit dosageforms are substantially homogeneous. As used herein, “homogeneous”refers to mixtures, solutions, suspensions, compositions, dosage forms,and/or formulations of the present disclosure that have a uniformdistribution of ingredients throughout. Homogeneity is synonymous withuniformity and can refer to intra-sample uniformity, batch-to-batchuniformity, run-to-run uniformity, and/or dosage form-to-dosage formuniformity. For example, intra-sample uniformity can be determined byanalyzing a first portion of a sample, mixture, or composition andcomparing this with a second portion of the same sample, mixture, orcomposition. Typical deviations of a composition (e.g., variation in thepercentage by weight of excipients and the like) of a substantiallyhomogeneous composition are about 5% or less, about 3% or less, about 2%or less, about 1% or less, or within experimental error.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a pharmaceutically acceptable excipient. As used herein, theterm “excipient” refers to any inert substance that can be combined witha nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., and the sulfoalkylether cyclodextrin for preparing the pharmaceutical compositions.

Pharmaceutically acceptable excipients suitable for use with the presentdisclosure include, but are not limited to, a carrier, a water-solublepolymer, a preservative, an antioxidant, a pH-adjusting agent (e.g., anacidifying agent, an alkalinizing agent, and/or a buffer), a bulkingagent, a complexation enhancing agent, a cryoprotectant, a densitymodifier, an electrolyte, a flavor, a fragrance, a lyophilizing aid(e.g., a bulking agent and/or stabilizing agent), a plasticizer, asolubility-enhancing agent, a stabilizing agent, a sweetener, a surfacetension modifier, a volatility modifier, a viscosity modifier, andcombinations thereof. In addition, one of skill in the art willrecognize that pharmaceutically acceptable excipients can be used in thepresent disclosure including those listed in The Handbook ofPharmaceutical Excipients, 5th Ed., The Pharmaceutical Press andAmerican Pharmacists Association, London, UK and Washington, D.C.(2006), which is incorporated herein by reference in its entirety.

In some embodiments, a composition comprising a nitrogen mustard, suchas melphalan, mechlorethamine, cyclophosphamide, ifosfamide,bendamustine, etc., and a water soluble cyclodextrin derivative, furthercomprises a bulking agent, such as mannitol, lactose, or a sugar.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a pharmaceutically acceptable carrier. As used herein, a“carrier” refers to a vehicle suitable for transferring and/or dilutinga pharmaceutical composition or unit dosage form. Pharmaceuticallyacceptable carriers suitable for use include, but are not limited to,liquids, solids, colloids, gels, and combinations thereof. Liquidcarriers suitable for use include solvents, liquid dispersion mediums,and the like, such as, but not limited to, water, ethanol, a polyol(e.g., glycerol, propylene glycol, liquid polyethylene glycols, and thelike), a vegetable oil, a nontoxic glyceryl ester, and combinationsthereof. In some embodiments, a liquid carrier is selected from: adextrose solution, a saline solution, plasma, and lactated Ringer'ssolution.

In some embodiments, a pharmaceutical composition or unit dosage formdoes not include propylene glycol. In some embodiments, a pharmaceuticalcomposition or unit dosage form does not include ethanol. In someembodiments, a pharmaceutical composition or unit dosage form does notinclude propylene glycol or ethanol.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a water-soluble polymer such as, but not limited to,homopolymers of N-polyvinylpyrrolidone (e.g., “povidone”), low molecularweight hydroxypropyl cellulose, low molecular weight methyl cellulose,low molecular weight hydroxypropyl methyl cellulose, and the like, andcombinations thereof.

In some embodiments, after dilution a cyclodextrin derivative is presentin the diluted pharmaceutical composition in a concentration of about 75mM, about 100 mM, or about 125 mM.

In some embodiments, after dilution a nitrogen mustard, such asmelphalan, mechlorethamine, cyclophosphamide, ifosfamide, bendamustine,etc., is present in the diluted pharmaceutical composition in aconcentration of 0.1 mg/mL to 50 mg/mL, 0.15 mg/mL to 40 mg/mL, 0.2mg/mL to 30 mg/mL, 0.3 mg/mL to 25 mg/mL, 0.4 mg/mL to 20 mg/mL, 0.45mg/mL to 15 mg/mL, 0.5 mg/mL to 10 mg/mL, about 0.45 mg/mL, about 1mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, or about 5 mg/mL.

In some embodiments, a pharmaceutical composition and/or a diluent foruse with a composition of the present disclosure is free of asolubilizing agent such as, but not limited to, water, an alcohol (e.g.,ethanol and the like), a polyol (e.g., glycerol, propylene glycol,liquid polyethylene glycols, and the like), a vegetable oil, a nontoxicglyceryl ester, and combinations thereof. Thus, in some embodiments thediluent consists essentially of water and optional tonicity-adjustingagents (e.g., 0.9% saline solution for injection, and the like).

In some embodiments, the pH of a pharmaceutical composition or unitdosage form is controlled. In some embodiments, a pharmaceuticalcomposition or unit dosage form comprises a pharmaceutically acceptablebuffer and/or pH adjusting agent (e.g., an acidifying agent and/oralkalinizing agent). In some embodiments, a pharmaceutical compositionor unit dosage form has a pH of about 4 to about 6, about 4 to about 5,about 5 to about 6, about 4, about 5, about 5.5, or about 6 afterdilution with an aqueous diluent.

In some embodiments, a pharmaceutical composition or unit dosage formthat is to be diluted prior to administration to a subject has a pH ofabout 2 to about 6, about 3 to about 6, about 4 to about 6, or about 5to about 6. In some embodiments, after dilution (e.g., with an liquidcarrier) a unit dosage form of the present disclosure has a pH of about4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about4.5, about 5, about 5.5, or about 6 at the time of administration to asubject in need thereof.

In some embodiments, a composition that is to be lyophilized has a pH ofabout 2 to about 6, about 3 to about 6, about 4 to about 6, or about 5to about 6. In some embodiments, after dilution (e.g., with an liquidcarrier) a unit dosage form of the present disclosure has a pH of about4 to about 6, about 4 to about 5, about 5 to about 6, about 4, about4.5, about 5, about 5.5, or about 6 at the time of administration to asubject in need thereof.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a buffer. In some embodiments, a pharmaceutical composition orunit dosage form comprises a buffer suitable to provide a dilutecomposition having a pH of about 4 to about 6, about 4 to about 5, about5 to about 6, about 4, about 4.5, about 5 about 5.5, or about 6. In someembodiments, a buffer is present in a concentration of about 0.01 M toabout 10 M, about 0.01 M to about 5 M, or about 0.01 M to about 1 M.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a pH-adjusting agent such as, but not limited to, anacidifying agent (e.g., citric acid, HCl, and the like), an alkalinizingagent (e.g., NaOH and the like), a salt form of an acid (e.g., sodiumcitrate and the like), and combinations thereof. In some embodiments, apharmaceutical composition or unit dosage form comprises a pH-adjustingagent in an amount sufficient to provide a dilute composition having apH of about 4 to about 6, about 4 to about 5, about 5 to about 6, about4, about 4.5, about 5 about 5.5, or about 6. In some embodiments, Insome embodiments, a pharmaceutical composition or unit dosage formcomprises sodium citrate in an amount of 50 mg to 500 mg, 75 mg to 400mg, 100 mg to 300 mg, 150 mg to 250 mg, or about 200 mg.

In some embodiments, a pharmaceutical composition or unit dosage formcomprises a second therapeutic agent. Suitable second therapeutic agentsinclude, but are not limited to, a platinum compound, an antimetabolite,a nitrosourea, a corticosteroid, a calcineurin inhibitor, a monoclonalantibody, a polyclonal antibody, a cytotoxic antibiotic, an interferon,an opioid, an antihistamine, a volume expander, a pressor agent, andcombinations thereof. Additional second therapeutic agents include, butare not limited to, doxorubicin, bortezomib, rituximab, thalidomide,lenalidomide, gemcitabine, thiotepa, fludarabine, carmustine, etoposide,cytarabine, granulocyte colony-stimulating factor, ADH-1, topotecan,palifermin, prednisone, arsenic trioxide, ascorbic acid, busulfan,buthionine sulfoximine, and combinations thereof.

As used herein, a “unit dosage form” refers to a composition containinga specific amount of a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc., thewhole of which is intended to be administered to a subject in a singledose. A unit dosage form can be distinguished from a supply of amulti-dose amount of a pharmaceutical composition, e.g., a bottle ofmedicine, from which a unit dose is measured out.

In some embodiments, a unit dosage form of the present disclosurecomprises a therapeutically effective amount of a nitrogen mustard, suchas melphalan, mechlorethamine, cyclophosphamide, ifosfamide,bendamustine, etc. As used herein, a “therapeutically effective amount”refers to an amount of a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc., thatelicits a biological or medicinal response in a tissue, system, animalor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician, which includes alleviation of the symptoms ofa disease or disorder being treated.

A unit dosage form typically comprises the pharmaceutical composition ofthe present disclosure and optionally, one or more pharmaceuticallyacceptable excipients, wherein the amount of a nitrogen mustard, such asmelphalan, mechlorethamine, cyclophosphamide, ifosfamide, bendamustine,etc., present in the unit dosage form is sufficient for a singleadministration to a subject in need thereof. Unit dosage forms include,but are not limited to, liquid solutions, liquid suspensions, liquiddispersions, emulsions, gels, powders, tablets, capsules, caplets, andthe like. Treatment of a disease or condition amenable to treatment witha nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc., can comprise periodicadministration of a unit dosage form of the present disclosure, forexample, once every two weeks, once every four weeks, or some otherinterval.

In some embodiments, a unit dosage form of the present disclosurecomprises 25 mg to 125 mg, or 150 mg to 250 mg of melphalan as ahydrochloride salt. In some embodiments, a unit dosage form of thepresent disclosure comprises 50 mg or 200 mg melphalan as ahydrochloride salt.

In some embodiments, a unit dosage form of the present disclosure is asolid. In some embodiments, a solid unit dosage form of the presentdisclosure is a lyophilized solid or an aseptic spray-dried solid. Insome embodiments, a dosage form of the present disclosure is suitablefor dilution and/or reconstitution with a predetermined amount of aliquid carrier. For example, a unit dosage form (e.g., a liquid or asolid) of the present disclosure can be diluted with 5 mL to 500 mL, 10mL to 100 mL, or 10 mL to 50 mL of a liquid carrier.

The pharmaceutical compositions and unit dosage forms are stable. Asused herein, stability can refer to either the shelf-life of anundiluted solid or liquid dosage form or the resistance to degradationof a diluted liquid dosage form. In particular, currently availablemelphalan compositions suitable for intravenous administration must beused as soon as possible after dilution due to the rapid degradation ofmelphalan in aqueous solution. However, the dosage forms describedherein are stable for a considerable time period after dilution, forexample, at least 90 minutes up to at least 48 hours or more. Thus, inthose embodiments in which a solid or liquid unit dosage form isdiluted, the diluting can be performed immediately prior toadministering, or sometime before the administering without anysignificant loss of therapeutic efficacy. This enables a liquidpharmaceutical composition or liquid unit dosage form of the presentdisclosure to be diluted 90 minutes to 48 hours in advance of use (i.e.,in advance of parenteral administration to a subject in need thereof).

In some embodiments, the melphalan in a pharmaceutical composition ofthe present disclosure degrades by 2% or less at about 25° C. within 5hours, or by 4% or less at about 25° C. within 10 hours after dilutionwith an aqueous diluent to provide a diluted composition comprising acyclodextrin derivative in a concentration of about 75 mM or about 125mM.

The primary degradation product of melphalan in aqueous solution ismelphalan monohydroxide (also known as monohydroxymelphalan), whichproceeds via a hydrolysis reaction. See, e.g., S. A. Stout et al., Int.J. Pharm. 24:193 (1985). In some embodiments, dilution of apharmaceutical composition of the present disclosure provides amelphalan monohydroxide concentration (based on a 100% initialconcentration of melphalan) of 2% or less within 5 hours of thediluting, when the diluted composition is maintained at room temperature(about 25° C.). In some embodiments, dilution of a pharmaceuticalcomposition of the present disclosure provides a melphalan monohydroxideconcentration (based on a 100% initial concentration of melphalan) of 4%or less within 10 hours of the diluting, when the diluted composition ismaintained at room temperature (about 25° C.). In some embodiments,dilution of a pharmaceutical composition of the present disclosureprovides a melphalan monohydroxide concentration (based on a 100%initial concentration of melphalan) of 2% or less within 24 hours of thediluting, or 4% or less within 48 hours of the diluting when the dilutedcomposition is maintained at a temperature of about 10° C. or less.

Furthermore, the pharmaceutical compositions of the present disclosurecan be stored prior to dilution for an extended period of time withoutany significant loss of melphalan. For example, a solid pharmaceuticalcomposition comprising melphalan and a cyclodextrin derivative contains2% or less, by weight, of a melphalan degradant after storage at 25° C.for a period of at least 2 years, or 5% or less, by weight, of amelphalan degradant after storage at 25° C. for a period of at least 3years.

In some embodiments, a dry powder pharmaceutical composition of thepresent disclosure forms 2% or less of melphalan monohydroxide (based ona 100% initial concentration of melphalan) after storage for 2 years atroom temperature.

Pharmaceutical Kits

The present disclosure is also directed to a pharmaceutical kitcomprising a first container comprising 25 mg to 125 mg of a nitrogenmustard, such as melphalan, as a hydrochloride salt and an optionalwater-soluble polymer, and a second container comprising an aqueousdiluent, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of at least 50:1 (w/w) relative to the a nitrogen mustard,such as melphalan, mechlorethamine, cyclophosphamide, ifosfamide,bendamustine, etc.; and wherein combining the first container and thesecond container provides a dilute pharmaceutical composition having apH of about 4 to about 6 that degrades by 2% or less at about 25° C.within 5 hours after the diluting.

The present disclosure is also directed to a pharmaceutical kitcomprising a first container comprising 150 mg to 250 mg of a nitrogenmustard, such as melphalan, as a hydrochloride salt and an optionalwater-soluble polymer; and a second container comprising an aqueousdiluent, an optional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆R₇, R₈ and R₉ is astraight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of 25:1 to 35:1 (w/w) relative to the a nitrogen mustard,such as melphalan, mechlorethamine, cyclophosphamide, ifosfamide,bendamustine, etc.; and wherein combining the first container and thesecond container provides a dilute pharmaceutical composition having apH of about 4 to about 6 that degrades by 2% or less at about 25° C.within 5 hours after the diluting.

Alternatively, a first container comprises a nitrogen mustard, such asmelphalan, mechlorethamine, cyclophosphamide, ifosfamide, bendamustine,etc., (in an amount described above) and a cyclodextrin derivative (asdescribed above), an optional water-soluble polymer (e.g., povidone andthe like), and an optional pH-adjusting agent; and a second containercomprises a diluent (e.g., water, saline, and the like), an optionaltonicity adjusting agent, and an optional pH-adjusting agent.

Materials suitable for use as the containers with the kits of thepresent disclosure include, but are not limited to, a glass (e.g.,borosilicate glass, amber glass, and the like), a plastic (e.g.,polypropylene, high-density polyethylene, poly(ethylene terephthalate,polystyrene, polycarbonate, and the like, and combinations thereof), ametal (e.g., a foil), and the like, and combinations thereof (e.g., aplastic-coated glass and/or metal).

Containers suitable for use with the pharmaceutical kits of the presentdisclosure include, but are not limited to, vials, bottles, sachets, andthe like. The containers can be opened and/or the contents can beremoved therefrom, by, for example, tearing, cutting, removing ascrew-top, removing a stopper, piercing, squeezing, and the like, andcombinations thereof.

In some embodiments, a first container comprises povidone in an amountof 10 mg to 30 mg, 15 mg to 25 mg, or about 20 mg. In some embodiments,a second container comprises a pH-adjusting agent (e.g., an acidifyingagent, an alkalinizing agent, and/or a buffer) in a concentrationsufficient to provide a pH of about 4 to about 6 when the firstcontainer and the second container are combined. In some embodiments, asecond container comprises sodium citrate in an amount of 50 mg to 500mg, 75 mg to 400 mg, 100 mg to 300 mg, 150 mg to 250 mg, or about 200mg.

In some embodiments, the cyclodextrin derivative present in the secondcontainer is a compound of formula II:

wherein 21-x of the R groups are H and x of the R groups are —(CH₂)₄⁻SO₃ ⁻Na⁺, and x=6.0-7.1; wherein the first container comprises about200 mg of a nitrogen mustard, such as melphalan, as a hydrochloridesalt; and wherein the cyclodextrin derivative is present in the secondcontainer in an amount of about 27:1, about 30:1, or about 32:1 (w/w)relative to the a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc.Methods of Administering and Treating

Some methods of treating a human being with a nitrogen mustard compound,such as melphalan, can comprise: administering a nitrogen mustardcomposition, such as a melphalan composition, intravenously to the humanbeing. For these methods a nitrogen mustard composition, such as amelphalan composition, can prepared by reconstituting any solidpharmaceutical composition comprising a nitrogen mustard and acyclodextrin derivative described herein using a pharmaceuticallyacceptable diluent. In some embodiments, the pharmaceutically acceptablediluent is normal saline.

A benefit of the method described in the paragraph above is thatreconstitution can be rapid. For example, reconstitution can beconducted in about 1 second to about 5 minutes, about 1 second to about2 minutes, or about 1 second to about 1 minute. Another advantage isthat reconstitution can be carried out some time before the compositionis administered to the human being. In some embodiments, thereconstituted composition is administered more than 1 hour, more than 2hours, more than 4 hours, up to about 20 hours, up to about 24 hours,about 4 hours to about 24 hours, about 8 hours to about 12 hours, about4 hours to about 8 hours, about 4 hours to about 20 hours, or about 8hours to about 20 hours after reconstitution occurs. Another advantageis that the melphalan composition does not need to be reconstituted by apharmacist.

In some embodiments, the human being that is being treated with anitrogen mustard compound is suffering from a neoplastic disorder, suchas multiple myeloma.

In some embodiments, the present disclosure is directed to methods ofdelivering a nitrogen mustard, such as melphalan, mechlorethamine,cyclophosphamide, ifosfamide, bendamustine, etc, to a subject in needthereof, the method comprising administering a pharmaceuticalcomposition or unit dosage form comprising a nitrogen mustard to thesubject in need thereof. The methods of the present disclosure includeparenteral administration of the pharmaceutical compositions or unitdosage forms.

In some embodiments, the pharmaceutical compositions or unit dosageforms (or diluted forms thereof) are intravenously administered.Intravenous administration includes, but is not limited to, a bolusinjection, an intravenous infusion, a limb perfusion, a normothermicisolated limb infusion, a percutaneous hepatic perfusion, and the like,and combinations thereof. Administering the compositions of the presentdisclosure can also be performed by injection and/or drip line using acannula, a central line, a peripherally inserted central catheter line,and the like.

In some embodiments, a pharmaceutical composition of the presentdisclosure is administered as an infusion for a duration of 15 minutesto 6 hours, 30 minutes to 4 hours, 45 minutes to 3 hours, 1 hour to 2hours, about 15 minutes, about 30 minutes, about 45 minutes, about 1hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours,about 4 hours, about 5 hours, or about 6 hours.

In some embodiments, the present disclosure is directed to parenterallyadministering a pharmaceutical composition or unit dosage form to asubject for which an oral composition of a nitrogen mustard, such asmelphalan, is for one or more reasons, not appropriate. For example,oral compositions of a nitrogen mustard may not be appropriate because asubject may be too young, unable to swallow, undergoing surgery,incapacitated, or have a disorder that blocks absorption of a nitrogenmustard administered via the oral route. Further, parenteraladministration of the pharmaceutical compositions of the presentdisclosure are useful for treating conditions in a subject in which arapid increase in the in vivo concentration of a nitrogen mustard isrequired.

In some embodiments, the present disclosure is directed to a method oftreating and or preventing diseases in a human subject by administeringthe pharmaceutical compositions and or unit dosage forms to the humansubject. In some embodiments, the present disclosure is directed tomethods of treating a subject suffering from a disease or disorderamenable to treatment with a nitrogen mustard, such as melphalan,mechlorethamine, cyclophosphamide, ifosfamide, bendamustine, etc. themethod comprising administering a pharmaceutical composition or unitdosage form comprising the nitrogen mustard to the subject. As usedherein the terms “treat,” “treating,” and “treatment” refer toadministering a composition of the present disclosure prior to the onsetof clinical symptoms of a disease state/condition so as to prevent thedevelopment of any symptom, as well as administering the compositionafter the onset of one or more clinical symptoms of a diseasestate/condition so as to reduce or eliminate any such symptom, aspect orcharacteristic of the disease state/condition. Such treating need not beabsolute to be useful. Additionally, the terms “treat” and “treatment”refer to both therapeutic treatment and prophylactic, maintenance, orpreventative measures, wherein the object is to prevent or slow down(lessen) an undesired physiological condition, disorder or disease, orobtain beneficial or desired clinical results. For purposes of thisdisclosure, beneficial or desired clinical results include, but are notlimited to, alleviation of a symptom or a sign; diminishment of extentof a condition, disorder or disease; stabilization (i.e., not worsening)of the state of condition, disorder or disease; delay in onset orslowing of condition, disorder or disease progression; amelioration of acondition, disorder or disease state, remission (whether partial ortotal), whether detectable or undetectable; or enhancement orimprovement of condition, disorder or disease. Treatment includeseliciting a clinically significant response, without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

As used herein, the term “subject” refers to warm blooded animals suchas mammals, including humans and non-humans, such as, but not limitedto, domestic and farm animals, zoo animals, sports animals, and pets(e.g., cats, dogs, mice, guinea pigs, horses, bovine cows, and sheep).In some embodiments, a subject is a human subject. Human subjectssuitable for administering the pharmaceutical compositions and unitdosage forms include, but are not limited to, pediatric, adult, andgeriatric subjects. In some embodiments of the disclosure, the subjectis a pediatric subject. For example, according to the U.S. Food and DrugAdministration, a “pediatric” subject is up to 21 years of age, andincludes neonates (birth to about 1 month of age), infants (about 1month to about 2 years of age), children (about 2 to about 12 years ofage) and adolescents (about 12 to about 21 years of age). See Guidancefor Industry and FDA Staff, Premarket Assessment of Pediatric MedicalDevices, U.S. Dept. of Health and Human Services, Food and DrugAdministration, Center for Devices and Radiological Health, and Centerfor Biologics Evaluation and Research (May 14, 2004). In someembodiments of the disclosure, the subject is an adult. As used herein,an “adult” subject is 18 years of age or older. In some embodiments, asubject is an adult that is about 50 years or older. In some embodimentsof the disclosure, the subject is geriatric. Geriatric subjects are atleast about 65 years of age. In some embodiments, a subject is about 70years of age or older.

In some embodiments, the subject is a pediatric subject suffering from adisorder such as, but not limited to, an inborn defect, animmunodeficiency, a combined immunodeficiency, a severe combinedimmunodeficiency, a congenital neutropenia with defective stem cells,aplastic anemia, and combinations thereof

In some embodiments, a subject is a geriatric subject scheduled toundergo a non-myeloablative procedure.

In some embodiments, the present disclosure comprises a method fortreating a subject who has or is at risk for developing a conditionamenable to treatment with melphalan, the method comprisingadministering an effective amount (i.e., a therapeutically effectiveamount) of a composition of the disclosure to the subject. Conditionsamenable to treatment with melphalan include, but are not limited to,neoplastic disorders.

In some embodiments, a therapeutically effective amount foradministering to a subject who has or is at risk for developing acondition amenable to treatment with melphalan is 25 mg to 125 mg, 40 mgto 110 mg, 40 mg to 75 mg, 40 mg to 60 mg, about 40 mg, about 50 mg,about 60 mg, about 75 mg, or about 100 mg of melphalan as ahydrochloride salt. The methods of the present disclosure also includetitrating upward or downward from an initial melphalan dose in order toprovide a therapeutically effective melphalan dosage. A therapeuticallyeffective dose can be administered once, twice, thrice, four times, fivetimes, six times, seven times, eight times, ten times, twelve times, ormore as needed.

In some embodiments, the present disclosure is directed to a method oftreating a disease, disorder or condition that is therapeuticallyresponsive to a stem cell transplantation, the method comprisingadministering to a subject in need thereof a pharmaceutical compositionor unit dosage form followed by the subject undergoing a stem celltransplantation.

In some embodiments, a method of the present disclosure comprisesadministering a pharmaceutical composition or unit dosage form (ordiluted form thereof) to a subject that suffers from a disorder selectedfrom: myeloma, multiple myeloma, acute myelogenous leukemia, malignantmelanoma, metastatic melanoma (e.g., metastatic ocular melanoma,metastatic cutaneous melanoma, and the like), breast cancer, ovariancancer, testicular cancer, advanced prostate cancer, a myelodysplasticsyndrome, a neuroendocrine cancer (e.g., a metastatic neuroendocrinetumor, and the like), a metastatic adenocarcinoma tumor, ahepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma,plasma cell neoplasm, amyloidosis, scleromyxedema, and combinationsthereof.

In some embodiments, a method of the present disclosure comprisesadministering a pharmaceutical composition or unit dosage form (ordiluted form thereof) to a subject for whom a stem cell transplantationhas been indicated (e.g., a hematopoietic stem cell transplantation). Insome embodiments, a subject for whom a stem cell transplantation hasbeen indicated suffers from a disease or disorder selected from: aleukemia, a cancer, a non-malignant disease, and combinations thereof.In some embodiments, a subject for whom a stem cell transplantation hasbeen indicated suffers from a disease or disorder selected from:myeloma, multiple myeloma, a lymphoma, non-Hodgkin lymphoma (“NHL”),leukemia, acute myeloid leukemia (“AML”), Hodgkin's disease, acutelymphoblastic leukemia (“ALL”), a myelodysplastic syndrome (“MDS”), amyeloproliferative disorder (“MPD”), chronic myelogenous leukemia(“CML”), neuroblastoma, aplastic anemia, chronic granulocytic leukemia,a neuroblastoma, sickle-cell disease, osteogenic sarcoma, Ewing'ssarcoma, a desmoplastic small round cell tumor, plasma cell neoplasm,amyloidosis, scleromyxedema, and combinations thereof. In someembodiments, a subject for whom a stem cell transplantation has beenindicated is a subject who would not benefit from prolonged treatmentwith, or is already resistant to, chemotherapy.

Thus, the pharmaceutical compositions and unit dosage forms are usefulfor treatment of a condition amenable to treatment with melphalan, aswell as use for conditioning a subject in need thereof for receiving astem cell transplantation.

The amount of the pharmaceutical composition that is administered istherapeutically effective for the treatment that is desired. Forexample, a therapeutically effective amount for the treatment ofmultiple myeloma refers to an amount which, when administered,diminishes one or more symptoms associated with this disorder.

In some embodiments, the present disclosure is directed to a method ofconditioning a subject in order to conduct a stem cell transplantation,the method comprising administering an effective amount of apharmaceutical composition or unit dosage of the disclosure (e.g.,intravenously) to the subject. Thus, the pharmaceutical compositions andunit dosage forms are useful for treating a subject who suffers from acondition amenable to treatment by a stem cell transplant. As usedherein, “stem cell transplantation” includes autologous and/or allogenictransplantation procedures.

The pharmaceutical compositions of the present disclosure are suitablefor administering a nitrogen mustard, such as melphalan, in a“high-intensity” or myeloblative conditioning regimen in preparation fora stem cell transplantation, or in a “reduced intensity” conditioningregimen in preparation for a stem cell transplantation. As used herein,“reduced intensity” conditioning refers to dosages in which a nitrogenmustard, such as melphalan, dose of less than 150 mg/m² is administeredto a subject at any one dose. In some embodiments, the pharmaceuticalcomposition is administered to a subject that is 50 years of age orolder who suffers from a condition amenable to treatment by a stem celltransplant.

In some embodiments, the melphalan is administered to a subject in needof a stem cell transplantation at a dose of 50 mg/m² to 300 mg/m², 50mg/m² to 250 mg/m², 50 mg/m² to 225 mg/m², 50 mg/m² to 200 mg/m², 50mg/m² to 175 mg/m², 50 mg/m² to 150 mg/m², 100 mg/m² to 300 mg/m², 100mg/m² to 250 mg/m², 100 mg/m² to 225 mg/m², 100 mg/m² to 200 mg/m², 100mg/m² to 175 mg/m², 100 mg/m² to 150 mg/m², 125 mg/m² to 300 mg/m², 125mg/m² to 250 mg/m², 125 mg/m² to 225 mg/m², 125 mg/m² to 200 mg/m², 150mg/m² to 300 mg/m², 150 mg/m² to 250 mg/m², 200 mg/m² to 300 mg/m², 200mg/m² to 250 mg/m², about 50 mg/m², about 100 mg/m², about 125 mg/m²,about 150 mg/m², about 175 mg/m², about 200 mg/m², about 250 mg/m², orabout 300 mg/m².

In some embodiments, the administering comprises a dosage administeredat four week intervals. In some embodiments, the dosage is administeredtwice, thrice, four times, five times, six times, eight times, or tentimes. For example, in some embodiments a dose of about 100 mg/m² isadministered three times with a four-week interval between the doses. Insome embodiments, a dose of about 200 mg/m² is administered twice with afour-week interval between the doses. The final dose can be followed bya stem cell transplantation.

The pharmaceutical compositions and unit dosage forms can beadministered alone or in conjunction with other medications orpharmaceutical compositions. In some embodiments, a method of thepresent disclosure comprises administering to a subject a secondtherapeutic agent selected from: an alkylating agent, a platinumcompound, an antimetabolite, a nitrosourea, a corticosteroid, acalcineurin inhibitor, a monoclonal antibody, a polyclonal antibody, acytotoxic antibiotic, an interferon, an opioid, an antihistamine, avolume expander, a pressor agent, and combinations thereof. Additionalsecond therapeutic agents include, but are not limited to, cisplatin,carboplatin, doxorubicin, bortezomib, rituximab, thalidomide,lenalidomide, gemcitabine, thiotepa, fludarabine, carmustine, etoposide,cytarabine, granulocyte colony-stimulating factor (G-CSF), ADH-1,topotecan, paliferm in, prednisone, arsenic trioxide, ascorbic acid,busulfan, cyclophosphamide, N,N′,N″-triethylenethiophosphoramide,buthionine sulfoximine, and combinations thereof. A second therapeuticagent can be administered to a subject either in a pharmaceuticalcomposition or unit dosage form that includes at least one additionaltherapeutic agent (in addition to a nitrogen mustard), or as a separatepharmaceutical composition or unit dosage.

In some embodiments, the pharmaceutical compositions and/or unit dosageforms are administered with other combinations of therapeutic activeagents such as, but not limited to, carmustine, etoposide andcytarabine; busulfan and thiotepa; doxorubicin and bortezomib; arsenictrioxide and citric acid; thalidomide and rituximab; thalidomide andprednisone; and busulfan, fludarabine and G-CSF.

In some embodiments, the pharmaceutical compositions and unit dosageforms can enhance the bioavailability, rate of therapeutic onset, and/ortherapeutic efficacy of a nitrogen mustard. Thus, the present disclosureis also directed to a method of decreasing the time to therapeutic onsetof a nitrogen mustard following administration thereof, the methodcomprising orally or parenterally administering to a subject in needthereof a pharmaceutical composition or unit dosage form, wherein thetime to therapeutic onset of a nitrogen mustard provided by the orallyor parenterally administered composition or unit dosage is less than thetime to therapeutic onset of a nitrogen mustard provided by an orallyadministered reference composition that excludes the cyclodextrinderivative and contains an equivalent dose of the nitrogen mustard. Insome embodiments, the time to therapeutic onset of the nitrogen mustardfollowing administration of a pharmaceutical composition or unit dosageform is reduced by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 40%, or at least 50% comparedto the time to therapeutic onset of the nitrogen mustard provided by anintravenously administered reference composition that excludes thecyclodextrin derivative and contains an equivalent dose of the nitrogenmustard.

In some embodiments, the dissolution of a nitrogen mustard from thedosage forms can be related to pharmacokinetic parameters and/or the invivo concentration of a nitrogen mustard and/or its metabolite(s). Thein vivo concentration of a nitrogen mustard and its metabolite(s), aswell as pharmacokinetic parameters associated with an active form of anitrogen mustard can be determined by, e.g., sampling the blood plasmaof a subject after administering a composition of the presentdisclosure. Pharmacokinetic parameters that can be measured include, butare not limited to, AUC_(0-t), AUC_(t-∞), AUC_(0-∞), and In(AUC_(LAST)).

As used herein, “AUC_(0-t)” refers to the Area Under the Concentrationtime curve (i.e., plot of plasma concentration vs. time) after nitrogenmustard administration. The area is conveniently determined by the“trapezoidal rule”: the data points are connected by straight linesegments, perpendiculars are erected from the abscissa to each datapoint, and the sum of the areas of the triangles and trapezoids soconstructed is computed.

As used herein, “AUC_(t-∞)” refers to the Area Under the Concentrationtime curve, wherein the last concentration is extrapolated to baselinebased on the rate constant for elimination.

As used herein, “AUC_(0-∞)” refers to the sum of the Area Under theConcentration time curves for AUC_(0-t) and AUC_(t-∞).

As used herein, “In(AUC_(LAST))” refers to the Area Under theConcentration time curve determined by plotting plasma concentration ona natural logarithmic scale, using the last measured plasmaconcentration as the end point.

As used herein, “IntraCV” refers to an intra-assay coefficient ofvariation, which is the standard deviation within a sample set dividedby the mean value of the sample set, with the result reported as apercentage.

In some embodiments, the bioavailability of melphalan in a human subjectfrom a composition of the present disclosure is substantially greaterthan that observed upon administration of an equivalent dose ofmelphalan from a formulation lacking a cyclodextrin derivative (e.g.,ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable(Bioniche Pharma USA)). For example, the dosage forms can have anAUC_(0-t) or AUC_(0-∞) that is at least 20% or greater, at least 25% orgreater, at least 30% or greater, at least 40% or greater, at least 50%or greater, at least 60% or greater, or at least 70% or greater than theAUC_(0-t) or AUC_(0-∞) observed after administration of a melphalanformulation to a subject that contains the same amount of melphalan andlacks a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).

In some embodiments, the bioavailability of melphalan from a compositionof the present disclosure is greater than that observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).For example, the dosage forms can have an AUC_(0-t) or AUC_(0-∞) that isat least 20% or greater, at least 25% or greater, at least 30% orgreater, at least 40% or greater, at least 50% or greater, at least 60%or greater, or at least 70% or greater than the AUC_(0-t) or AUC_(0-∞)observed after administration of a melphalan formulation to a subjectthat contains the same amount of melphalan and lacks a cyclodextrinderivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or MelphalanHCl Injectable (Bioniche Pharma USA)). In some embodiments, theAUC_(0-t) or AUC_(0-∞) of melphalan from a composition of the presentdisclosure is 20% to 70%, 20% to 60%, 20% to 50%, 30% to 70%, 30% to60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50% to 70% greater than theAUC_(0-t) or AUC_(0-∞) observed upon administration of an equivalentdose of melphalan from a formulation lacking a cyclodextrin derivative(e.g., ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HClInjectable (Bioniche Pharma USA)).

In some embodiments, the maximum plasma concentration (C_(max)) ofmelphalan from a composition of the present disclosure is at least 20%or greater, at least 25% or greater, at least 30% or greater, at least40% or greater, at least 50% or greater, at least 60% or greater, or atleast 70% or greater than a C_(max) observed upon administration of anequivalent dose of melphalan from a formulation lacking a cyclodextrinderivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or MelphalanHCl Injectable (Bioniche Pharma USA)). In some embodiments, the maximumplasma concentration (C_(max)) of melphalan from a composition of thepresent disclosure is 20% to 70%, 20% to 60%, 20% to 50%, 30% to 70%,30% to 60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50% to 70% greaterthan a C_(max) observed upon administration of an equivalent dose ofmelphalan from a formulation lacking a cyclodextrin derivative (e.g.,ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable(Bioniche Pharma USA)).

In some embodiments, the rate of therapeutic onset of melphalan from acomposition of the present disclosure is faster than that observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).For example, the dosage forms have a time to C_(max) (i.e., t_(max))that is about 5%, about 10%, about 15%, about 20%, about 25%, or about30% faster, or about 35% faster than a t_(max) observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). Insome embodiments, the dosage forms have a time to C_(max). (i.e.,t_(max)) that is 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 10% to 35%,15% to 35%, 20% to 35%, or 25% to 30% faster than a t_(max) observedupon administration of an equivalent dose of melphalan from aformulation lacking a cyclodextrin derivative (e.g., ALKERAN® forInjection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche PharmaUSA)).

In some embodiments, the pharmaceutical compositions of the presentdisclosure provide a reduced rate of hypersensitivity in patients afterparenteral administration compared with patients parenterallyadministered a similar dose of melphalan without a cyclodextrinderivative.

In some embodiments, the pharmaceutical compositions of the presentdisclosure provide a reduced rate of severe myelotoxicity in patients(e.g., patients who experience a white blood cell count <1,000 per mLand/or platelet count <25,000) after parenteral administration comparedwith patients parenterally administered a similar dose of melphalanwithout a cyclodextrin derivative.

In some embodiments, the pharmaceutical compositions of the presentdisclosure provide a reduced rate of death in patients after parenteraladministration compared with patients parenterally administered asimilar dose of melphalan without a cyclodextrin derivative.

Having generally described the disclosure, a further understanding canbe obtained by reference to the examples provided herein. These examplesare given for purposes of illustration only and are not intended to belimiting.

The following is a listing of embodiments that are specificallycontemplated herein.

-   -   Embodiment 1. A method of treating a subject suffering from a        neoplastic disorder, the method comprising:        -   diluting a composition with an aqueous diluent to provide a            dilute pharmaceutical composition comprising 25 mg to 125 mg            of melphalan and a cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃—            group;        -   wherein the dilute pharmaceutical composition has a pH of            about 4 to about 6; wherein the cyclodextrin derivative is            present in a concentration of at least 50:1 (w/w) relative            to the melphalan;        -   wherein the melphalan in the dilute pharmaceutical            composition degrades by 2% or less at about 25° C. within 5            hours after the diluting; and        -   administering the dilute pharmaceutical composition by            injection to the subject in need thereof.

    -   Embodiment 2. The method of embodiment 1, wherein the neoplastic        disorder is selected from: myeloma, multiple myeloma, melanoma,        acute myelogenous leukemia, malignant melanoma, breast cancer,        ovarian cancer, testicular cancer, advanced prostate cancer, a        neuroendocrine cancer, metastatic melanoma, a metastatic        neuroendocrine tumor, a metastatic adenocarcinoma tumor,        hepatocellular carcinoma, osteogenic sarcoma, polycythemia        veraplasma, plasma cell neoplasm, amyloidosis, scleromyxedema,        and combinations thereof.

    -   Embodiment 3. The method of embodiment 2, wherein the neoplastic        disorder is multiple myeloma and the administering is systemic        and provides palliative treatment of the multiple myeloma.

    -   Embodiment 4. The method of embodiment 1, wherein at least one        of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a        hydroxy-substituted-C₃ group.

    -   Embodiment 5. The method of embodiment 1, wherein R₁, R₂, R₃,        R₄, R₅, R₆, R₇, R₈ and R₉ are independently a straight-chain or        branched C₁-C₈-(alkylene)-SO₃ ⁻ group having a degree of        substitution of 4 to 8 per cyclodextrin derivative, and the        remaining substituents are —H.

    -   Embodiment 6. The method of embodiment 1, wherein at least one        of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is substituted with a        straight-chain C₄-(alkylene)-SO₃ ⁻ group.

    -   Embodiment 7. The method of embodiment 1, wherein the        cyclodextrin derivative is a compound of formula II:

-   -   -   wherein 21-x of the R groups are H and x of the R groups are            —(CH₂)₄ ⁻SO₃ ⁻Na⁺, and x=6.0-7.1;        -   wherein the pharmaceutical composition comprises about 50 mg            of melphalan as a hydrochloride salt; and        -   wherein the cyclodextrin derivative is present in a            concentration of 50:1 to 100:1 (w/w) relative to the            melphalan.

    -   Embodiment 8. The method of embodiment 1, wherein the dilute        pharmaceutical composition is substantially free of an alcohol.

    -   Embodiment 9. The method of embodiment 1, wherein the aqueous        diluent is a saline solution.

    -   Embodiment 10. The method of embodiment 1, wherein the subject        suffering from the neoplastic disorder is a pediatric subject.

    -   Embodiment 11. The method of embodiment 1, wherein the melphalan        in the dilute pharmaceutical composition degrades by 4% or less        at 25° C. within 10 hours after the diluting.

    -   Embodiment 12. The method of embodiment 1, wherein the dilute        pharmaceutical composition is stored about 0.5 hours to about 18        hours prior to the administering.

    -   Embodiment 13. The method of embodiment 1, wherein the        administering provides a melphalan C_(max) in the subject        suffering from a neoplastic disorder that is at least 20% or        greater than a melphalan C_(max) provided by a melphalan        formulation containing an equivalent dose of melphalan and        lacking the cyclodextrin derivative.

    -   Embodiment 14. The method of embodiment 1, wherein the        administering provides a melphalan AUC_(0-t) in the subject        suffering from a neoplastic disorder that is at least 20% or        greater than a melphalan AUC_(0-t) provided by a melphalan        formulation containing an equivalent dose of melphalan and        lacking the cyclodextrin derivative.

    -   Embodiment 15. A method for conditioning a subject for whom a        stem cell transplantation has been indicated, the method        comprising:        -   administering a melphalan dose of 50 mg/m² to 300 mg/m² per            day to the subject for whom a stem cell transplantation has            been indicated, wherein the melphalan dose is administered            in a pharmaceutical composition comprising melphalan and a            cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻            group;        -   wherein the pharmaceutical composition has a pH of about 4            to about 6; and        -   wherein the cyclodextrin derivative is present in a ratio of            at least 25:1 (w/w) relative to the melphalan.

    -   Embodiment 16. The method of embodiment 15, wherein the        administering is for a period of two or more days.

    -   Embodiment 17. The method of embodiment 15, wherein the subject        in need of the stem cell transplantation is a pediatric subject.

    -   Embodiment 18. The method of embodiment 15, wherein the        administering is performed intravenously.

    -   Embodiment 19. The method of embodiment 15, wherein the        administering is performed via a limb perfusion.

    -   Embodiment 20. The method of embodiment 15, wherein at least one        of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a        hydroxy-substituted-C₃ group.

    -   Embodiment 21. The method of embodiment 15, wherein the        cyclodextrin derivative is a compound of formula

-   -   -   wherein 21-x of the R groups are H and x of the R groups are            —(CH₂)₄ ⁻SO₃ ⁻ Na⁺, and x=6.0-7.1;        -   wherein the pharmaceutical composition comprises about 200            mg of melphalan as a hydrochloride salt; and        -   wherein the cyclodextrin derivative is present in a ratio of            25:1 to 35:1 (w/w) relative to the melphalan.

    -   Embodiment 22. The method of embodiment 15, wherein the subject        for whom a stem cell transplantation has been indicated suffers        from a disease or disorder selected from: myeloma, multiple        myeloma, a lymphoma, non-Hodgkin lymphoma, leukemia, acute        myeloid leukemia, Hodgkin's disease, acute lymphoblastic        leukemia, a myelodysplastic syndrome, a myeloproliferative        disorder, chronic myelogenous leukemia, neuroblastoma, aplastic        anemia, chronic granulocytic leukemia, a neuroblastoma,        sickle-cell disease, osteogenic sarcoma, Ewing's sarcoma, a        desmoplastic small round cell tumor, plasma cell neoplasm,        amyloidosis, scleromyxedema, and combinations thereof.

    -   Embodiment 23. The method of embodiment 15, comprising diluting        a concentrated melphalan composition with an aqueous diluent to        provide the pharmaceutical composition.

    -   Embodiment 24. The method of embodiment 23, wherein the        concentrated melphalan composition comprises 50 mg to 500 mg of        melphalan.

    -   Embodiment 25. The method of embodiment 23, wherein the        concentrated melphalan composition comprises about 200 mg.

    -   Embodiment 26. The method of embodiment 15, wherein the        pharmaceutical composition is substantially free of an alcohol.

    -   Embodiment 27. The method of embodiment 23, wherein the aqueous        diluent is a saline solution.

    -   Embodiment 28. The method of embodiment 23, wherein the        melphalan in the pharmaceutical composition degrades by 4% or        less at about 25° C. within 10 hours after the diluting.

    -   Embodiment 29. The method of embodiment 23, wherein the        pharmaceutical composition is stored about 0.5 hours to about 12        hours prior to the administering.

    -   Embodiment 30. The method of embodiment 15, wherein the        administering provides a melphalan C_(max) in the subject for        whom a stem cell transplantation has been indicated that is at        least 20% or greater than a melphalan C_(max) provided by a        melphalan formulation containing an equivalent dose of melphalan        and lacking the cyclodextrin derivative.

    -   Embodiment 31. The method of embodiment 15, wherein the        administering provides a melphalan AUC_(0-t) in the subject for        whom a stem cell transplantation has been indicated that is at        least 20% or greater than a melphalan AUC_(0-t) provided by a        melphalan formulation containing an equivalent dose of melphalan        and lacking the cyclodextrin derivative.

    -   Embodiment 32. A pharmaceutical composition comprising:

    -   25 mg to 125 mg of melphalan as a hydrochloride salt;

    -   an optional buffer; and

    -   a cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻            group;        -   wherein the pharmaceutical composition has a pH of about 4            to about 6,        -   wherein dilution of the pharmaceutical composition with an            aqueous solution provides a dilute pharmaceutical            composition in which the melphalan degrades by 2% or less at            about 25° C. within 5 hours after the dilution; and        -   wherein the cyclodextrin derivative is present in a ratio of            50:1 to 100:1 (w/w) relative to the melphalan.

    -   Embodiment 33. The pharmaceutical composition of embodiment 32,        wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is        a hydroxy-substituted-C₃ group.

    -   Embodiment 34. The pharmaceutical composition of embodiment 32,        wherein the cyclodextrin derivative is a compound of formula II:

-   -   -   wherein 21-x of the R groups are H and x of the R groups are            —(CH₂)₄ ⁻SO₃ ⁻Na⁺, and x=6.0-7.1;        -   wherein the pharmaceutical composition comprises about 50 mg            of melphalan as a hydrochloride salt; and        -   wherein the cyclodextrin derivative is present in a ratio of            about 55:1 (w/w) relative to the melphalan.

    -   Embodiment 35. A pharmaceutical composition comprising:

    -   150 mg to 250 mg of melphalan as a hydrochloride salt;

    -   an optional buffer; and

    -   a cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻            group;        -   wherein the pharmaceutical composition has a pH of about 4            to about 6,        -   wherein dilution of the pharmaceutical composition with an            aqueous solution provides a melphalan solution in which the            melphalan degrades by 2% or less at about 25° C. within 5            hours after the dilution; and        -   wherein the cyclodextrin derivative is present in a ratio of            25:1 to 35:1 (w/w) relative to the melphalan.

    -   Embodiment 36. The pharmaceutical composition of embodiment 35,        wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is        a hydroxy-substituted-C₃ group.

    -   Embodiment 37. The pharmaceutical composition of embodiment 35,        wherein the cyclodextrin derivative is a compound of formula II:

-   -   -   wherein 21-x of the R groups are H and x of the R groups are            —(CH₂)₄ ⁻SO₃ ⁻Na⁺, and x=6.0-7.1;        -   wherein the pharmaceutical composition comprises about 200            mg of melphalan as a hydrochloride salt; and        -   wherein the cyclodextrin derivative is present in a ratio of            about 30:1 (w/w) relative to the melphalan.

    -   Embodiment 38. A pharmaceutical kit comprising:        -   a first container comprising 25 mg to 125 mg of melphalan as            a hydrochloride salt and an optional water-soluble polymer;            and        -   a second container comprising an aqueous diluent, an            optional buffer, and a cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻            group;        -   wherein the cyclodextrin derivative is present in the second            container in a concentration of at least 50:1 (w/w) relative            to the melphalan; and        -   wherein combining the first container and the second            container provides a dilute pharmaceutical composition            having a pH of about 4 to about 6 that degrades by 2% or            less at about 25° C. within 5 hours after the diluting.

    -   Embodiment 39. The pharmaceutical kit of embodiment 38, wherein        the first container comprises povidone in an amount of 10 mg to        30 mg, and the second container comprises a pH-adjusting agent        in a concentration sufficient to provide a pH of about 4 to        about 6 when the first container and the second container are        combined.

    -   Embodiment 40. A pharmaceutical kit comprising:        -   a first container comprising 150 mg to 250 mg of melphalan            as a hydrochloride salt and an optional water-soluble            polymer; and        -   a second container comprising an aqueous diluent, an            optional buffer, and a cyclodextrin derivative of formula I:

-   -   -   wherein n is 4, 5 or 6;        -   wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are            independently —H, a straight-chain or branched            C₁-C₈-(alkylene)-SO₃ ⁻ group, or an optionally substituted            straight-chain or branched C₁-C₆ group;        -   wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and            R₉ is a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻            group;        -   wherein the cyclodextrin derivative is present in the second            container in a concentration of 25:1 to 35:1 (w/w) relative            to the melphalan; and        -   wherein combining the first container and the second            container provides a dilute pharmaceutical composition            having a pH of about 4 to about 6 that degrades by 2% or            less at about 25° C. within 5 hours after the diluting.

    -   Embodiment 41. The pharmaceutical kit of embodiment 40, wherein        the first container comprises povidone in an amount of 10 mg to        30 mg, and the second container comprises a pH-adjusting agent        in a concentration sufficient to provide a pH of about 4 to        about 6 when the first container and the second container are        combined.

    -   Embodiment 42. The pharmaceutical kits of embodiment 40, wherein        the cyclodextrin derivative is a compound of formula II:

-   -   -   wherein 21-x of the R groups are H and x of the R groups are            —(CH₂)₄ ⁻SO₃ ⁻ Na⁺, and x=6.0-7.1;        -   wherein the first container comprises about 200 mg of            melphalan as a hydrochloride salt; and        -   wherein the cyclodextrin derivative is present in the second            container in an amount of about 30:1 (w/w) relative to the            melphalan.

    -   Embodiment 43. A pharmaceutical product comprising a nitrogen        mustard compound and a water-soluble cyclodextrin derivative in        a single vial, wherein the product does not include propylene        glycol or ethanol.

    -   Embodiment 44. The pharmaceutical product of embodiment 43,        wherein the nitrogen mustard compound is melphalan.

    -   Embodiment 45. The pharmaceutical product of embodiment 44,        wherein the water-soluble cyclodextrin derivative and melphalan        have a weight ratio of at least about 50:1.

    -   Embodiment 46. The pharmaceutical product of embodiment 44 or        45, wherein melphalan and the water-soluble cyclodextrin are        present in a solid.

    -   Embodiment 47. The pharmaceutical product of embodiment 44, 45,        or 46, wherein the vial contains about 50 mg of melphalan.

    -   Embodiment 48. The pharmaceutical product of embodiment 43, 44,        45, 46, or 47, wherein the vial contains about 2500 mg to about        3000 mg of the water-soluble cyclodextrin derivative.

    -   Embodiment 49. The pharmaceutical product of embodiment 43, 44,        45, 46, 47, or 48, wherein the water-soluble cyclodextrin        derivative is an (SBE)-β-cyclodextrin.

    -   Embodiment 50. The pharmaceutical product of embodiment 43, 44,        45, 46, 47, 48, or 49, wherein the water-soluble cyclodextrin        derivative is (SBE)_(6.5 m)-β-cyclodextrin.

    -   Embodiment 51. A method of treating a human being with melphalan        comprising:        -   administering a melphalan composition intravenously to the            human being;        -   wherein the melphalan composition is prepared by            reconstituting contents of the vial of the pharmaceutical            product according to embodiment 43, 44, 45, 46, 47, 48, 49,            or 50 using a pharmaceutically acceptable diluent;        -   wherein the reconstituting is conducted in about 1 second to            about 5 minutes; and        -   wherein the melphalan composition is administered more than            1 hour after the melphalan composition is reconstituted.

    -   Embodiment 52. The method of embodiment 51, wherein the        pharmaceutically acceptable diluent is normal saline.

    -   Embodiment 53. The method of embodiment 51 or 52, wherein the        melphalan composition is administered up to about 24 hours after        the melphalan composition is reconstituted.

    -   Embodiment 54. The method of embodiment 51 or 52, wherein the        melphalan composition is administered about 4 hours to about 24        hours after the melphalan composition is reconstituted.

    -   Embodiment 55. The method of embodiment 51 or 52, wherein the        melphalan composition is administered about 8 hours to about 20        hours after the melphalan composition is reconstituted.

    -   Embodiment 56. The method of embodiment 51, 52, 53, 54, or 55,        wherein the melphalan composition is not reconstituted by a        pharmacist.

    -   Embodiment 57. The method of embodiment 51, 52, 53, 54, 55, or        56, wherein the melphalan composition has a pH of about 5 to        about 5.5.

    -   Embodiment 58. The method of embodiment 51, 52, 53, 54, 55, 56,        or 57, wherein the human being is suffering from a neoplastic        disorder.

    -   Embodiment 59. The method of embodiment 58, wherein the human        being is suffering from multiple myeloma.

    -   Embodiment 60. A pharmaceutical product comprising a nitrogen        mustard compound and a water-soluble cyclodextrin derivative in        a single vial, wherein the product does not include propylene        glycol or ethanol.

    -   Embodiment 61. The pharmaceutical product of embodiment 60,        wherein the nitrogen mustard compound is melphalan.

    -   Embodiment 62. The pharmaceutical product of embodiment 61,        wherein the water-soluble cyclodextrin derivative and melphalan        have a weight ratio of at least about 50:1.

    -   Embodiment 63. The pharmaceutical product of embodiment 61 or        62, wherein melphalan and the water-soluble cyclodextrin are        present in a solid.

    -   Embodiment 64. The pharmaceutical product of embodiment 61, 62,        or 63, wherein the vial contains about 50 mg of melphalan.

    -   Embodiment 65. The pharmaceutical product of embodiment 61, 62,        63, or 64, wherein the vial contains about 2500 mg to about 3000        mg of the water-soluble cyclodextrin derivative.

    -   Embodiment 66. The pharmaceutical product of embodiment 61, 62,        63, 64, or 65, wherein the water-soluble cyclodextrin derivative        is an (SBE)-β-cyclodextrin.

    -   Embodiment 67. The pharmaceutical product of embodiment 61, 62,        63, 64, 65, or 66, wherein the water-soluble cyclodextrin        derivative is (SBE)_(6.5 m)-β-cyclodextrin.

    -   Embodiment 68. A method of treating a human being with melphalan        comprising:        -   administering a melphalan composition intravenously to the            human being;        -   wherein the melphalan composition is prepared by            reconstituting contents of the vial of the pharmaceutical            product according to any one of embodiments 60-67 using a            pharmaceutically acceptable diluent;        -   wherein the reconstituting is conducted in about 1 second to            about 5 minutes; and        -   wherein the melphalan composition is administered more than            1 hour after the melphalan composition is reconstituted.

    -   Embodiment 69. The method of embodiment 68, wherein the        pharmaceutically acceptable diluent is normal saline.

    -   Embodiment 70. The method of embodiment 68 or 69, wherein the        melphalan composition is administered up to about 24 hours after        the melphalan composition is reconstituted.

    -   Embodiment 71. The method of embodiment 68 or 69, wherein the        melphalan composition is administered about 4 hours to about 24        hours after the melphalan composition is reconstituted.

    -   Embodiment 72. The method of embodiment 68 or 69, wherein the        melphalan composition is administered about 8 hours to about 20        hours after the melphalan composition is reconstituted.

    -   Embodiment 73. The method of any of embodiments 68-72, wherein        the melphalan composition is not reconstituted by a pharmacist.

    -   Embodiment 74. The method of any of embodiments 68-73 wherein        the melphalan composition has a pH of about 5 to about 5.5.

    -   Embodiment 75. The method of any of embodiments 68-74, wherein        the human being is suffering from a neoplastic disorder.

    -   Embodiment 76. The method of embodiment 75, wherein the human        being is suffering from multiple myeloma.

EXAMPLES Example 1

The dissolution rate of free-base melphalan (Chemwerth, Woodbridge,Conn.) in solutions at various pH and at various concentrations of acyclodextrin derivative were examined. The procedure was as follows:free base melphalan was added to a solution containing a cyclodextrinderivative (SBE_(6.5)-β-CD, CAPTISOL®) and then vortex mixed for 1-5minutes, and, if necessary, sonicated in ice water until a clearsolution was achieved.

TABLE Dissolution times for free base melphalan as a function ofcyclodextrin derivative concentration, volume, and pH. TargetDissolution Melphalan SBE_(6.5)-β-CD Time Conc. Conc. Volume pH (min) 50mg/mL 200 mM 5 mL 5 50 50 mg/mL 125 mM 6 mL 5 90 50 mg/mL 100 mM 7 mL 5160 50 mg/mL 75 mM 8 mL 5 >180 50 mg/mL 50 mM 10 mL  5 360 50 mg/mL 125mM 6 mL 2.7 75 50 mg/mL 125 mM 10 mL  1.8 16 50 mg/mL 125 mM 6 mL 1.3 550 mg/mL 75, 100 125 mM 10 mL  1.1 <5 for all

Referring to the data in the above Table, the dissolution of free basemelphalan was very rapid at pH 1.1 regardless of the concentration ofthe cyclodextrin derivative. After dissolution, the solution was thenneutralized with sodium hydroxide.

The solutions of free base melphalan in this example can be prepared byaddition of free base melphalan to a solution that contains thecyclodextrin derivative, or by adding a 0.1 M HCl solution to themelphalan and then adding the cyclodextrin derivative, or dissolving thefree base melphalan and cyclodextrin derivative simultaneously. However,sonication was superior to mixing and/or shaking for dissolutionenhancement and de-clumping of dry material in solution.

Example 2

The binding of free-base melphalan (Chemwerth, Woodbridge, Conn.) wasstudied as a function of cyclodextrin derivative concentration at pH 5and pH 7, and the data was compared with literature reports of free-basemelphalan binding. The pH 5 solutions contained 100 mM sodium bitartratebuffer, and was adjusted to pH 5 using sodium hydroxide in 0.9% sodiumchloride solution. The pH 7 solutions contained 50 mM each of mono- anddi-basic phosphate and 0.9% sodium chloride. Solutions containing 0, 50,75 and 100 mM SBE_(6.5)-β-CD (CAPTISOL®) were prepared, and excess freebase melphalan was added to 2 mL samples of each solution. After theaddition of free base melphalan, the samples were vortex mixed for 30seconds, sonicated in ice water bath for 20 minutes, and then mixed byend-over-end rotation at room temperature for 30 minutes. The sampleswere then centrifuged, the clear supernatant was diluted with water, andanalyzed by HPLC.

All melphalan assays performed by HPLC utilized the following protocol.A Shimadzu HPLC equipped with a SCL-10A system controller, SIL-10A autoinjector, LC-10AT liquid chromatograph, SPD-10A UV spectrophotometerdetector, CTO-10A column oven, and Class-VP chromatography laboratoryautomated software was utilized. The column was a ZORBAX® RX-C18 4.6 mmby 150 mm column (Agilent Technologies) having a 5 μm particle size.Samples were injected (20 μL) onto the column for isocratic elutionusing mobile phase of phosphate buffered saline (pH7.4):methanol:glacial acetic acid in a ratio of 500:250:10 (v/v). Themobile phase was selected in order to decrease or nominally quenchmelphalan conversion by having a high-chloride concentration. Thesamples were prepared immediately prior to injection. Detection was at260 nm.

The literature procedure involved adding an excess amount of melphalanto 0, 10, 20, 30, 40, 50, 75 and 100 mM solutions of SBE₇-β-CD (avg.M.W.=2248 g/mol) in a 25 mM phosphate buffer solution at pH 7.5. Thesuspensions were placed in tightly capped vials, sonicated 1 h, andagitated at 25° C. for 23 h. The solutions were then centrifuged, theclear supernatant was diluted with doubly distilled water, and analyzedby HPLC. See D. Q. Ma et al., J. Pharm. Sci. 89:275 (1999).

The data from the free base melphalan solubilization studies areprovided in FIG. 1. Referring to FIG. 1, free base melphalan displayed asignificantly lower solubility enhancement than that provided in aprevious literature report. See id. Because the solubility enhancementof free base melphalan provided by SBE_(6.5)-β-CD was lower thanexpected, additional phase solubility tests were performed usingmelphalan hydrochloride.

Example 3

The binding of melphalan hydrochloride (USP reference standard) and freebase melphalan (Chemwerth) with a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol) was determined as afunction of cyclodextrin derivative concentration at pH 7.5. Thetemperature was maintained at 22° C., and a 25 mM phosphate buffer wasadded to each solution. The data was compared with a literature reportof free-base melphalan binding with SBE₇-β-CD (avg. M.W.=2248 g/mol) ina 25 mM phosphate buffer at pH 7.5 (see Example 2).

The samples were prepared by adding excess melphalan hydrochloride orfree base melphalan to a 1 mL sample of various SBE_(6.5)-(β-CDsolutions. The samples were vortex mixed for 30 seconds, sonicated at20-24° C. for 60 minutes, and then mixed by end-over-end rotation at 22°C. for 60 minutes. The samples were then centrifuged, the clearsupernatant was diluted with water, and analyzed by HPLC. The data areprovided in FIG. 2. Referring to FIG. 2, the melphalan hydrochloridesalt displayed a significant solubility enhancement compared to freebase melphalan for all cyclodextrin derivative concentrations above 25mM.

Example 4

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process outlined schematically in FIG. 3.Referring to FIG. 3, water for injection, USP was placed in a stainlesssteel mixer at a temperature of 15-20° C., and hydrochloric acid wasadded until a pH of about 4.6 was achieved. The resulting solution wasstirred at a speed sufficient to produce a vortex (but without foamingor frothing) for about 15 minutes, a cyclodextrin derivative (27.2 gSBE_(6.5)-β-CD, CAPTISOL®) was added slowly while vortex stirring, andthe resulting solution was stirred for about 15 minutes to ensurecomplete dissolution. The resulting solution had a pH of about 2.5.Melphalan as a hydrochloride salt (516 mg) was added slowly while vortexstirring, and the resulting solution was stirred for about 15 minutes toensure complete dissolution. A base (2 N NaOH) was then slowly addedwhile vortex stirring until the solution had a pH of about 5.6. Thesolution was then assayed using a UV/vis spectrophotometer (detectionwavelength of 260 nm). The solution comprised melphalan at aconcentration of 5.16 mg/mL, and the melphalan was present in a ratio ofabout 1:55 w/w relative to the cyclodextrin derivative. The solution wasthen passed through a sterile filter (0.22 μm PVDF) and cooled to 10-15°C.

Example 5

The liquid pharmaceutical composition provided in Example 4 waslyophilized to provide a reconstitutable and/or dilutable dry powdercomprising 50 mg of melphalan as a hydrochloride salt. Glass vials werefilled with the solution (10 mL) and placed in trays on a pre-cooledshelf at 5° C. The vials were allowed to thermally equilibrate for about30 minutes, and were then lyophilized to provide a dry powder in eachvial. The vials were back-filled with nitrogen at a pressure of about400 mTorr, and then sealed.

Example 6

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process described in Example 4, and outlinedschematically in FIG. 3, except that the final solution containedmelphalan at a concentration of 10 mg/mL, and the melphalan was presentin a ratio of about 1:27 w/w relative to the cyclodextrin derivative.

Example 7

The liquid pharmaceutical composition provided in Example 6 waslyophilized to provide a reconstitutable and/or dilutable dry powdercomprising 200 mg of melphalan as a hydrochloride salt. Glass vials werefilled with the solution (20 mL) and placed in trays on a pre-cooledshelf at 5° C. The vials were allowed to thermally equilibrate for about30 minutes, and were then lyophilized to provide a dry powder in eachvial. The vials were back-filled with nitrogen at a pressure of about400 mTorr, and then sealed, packaged, and labeled. The vials wereprotected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Prophetic Example A

The liquid pharmaceutical composition provided in Examples 4 and 6 willbe aseptically spray dried to provide a free-flowing powder to be filledaseptically. The free-flowing powder will meet or exceed the dissolutionproperties of the lyophilized powder prepared in Examples 4 or 6.

Example 8

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process outlined schematically in FIG. 4.Referring to FIG. 4, water for injection, USP was placed in a stainlesssteel mixer at a temperature of 18-25° C., and the resulting solutionwas stirred at a speed sufficient to produce a vortex (but withoutfoaming or frothing). A cyclodextrin derivative (SBE_(6.5)-β-CD,CAPTISOL®) was added slowly while vortex stirring, and the resultingsolution was stirred for about 15 minutes to ensure completedissolution. The resulting solution was then cooled to about 2-8° C.Melphalan as a hydrochloride salt was added slowly while vortexstirring, and the resulting solution was stirred for about 15 minutes toensure complete dissolution. A base (2 N NaOH) was then slowly addedwhile vortex stirring until the solution had a pH of about 5-6 (targetpH 5.5). An in-process control (“IPC”) assay was then performed tomonitor pH, and the solution was diluted to the final target volumeusing water for injection, USP. The solution was then assayed using aUV/vis spectrophotometer (detection wavelength of 260 nm) and abioburden assay was performed. The solution was then passed through asterile filter (0.22 μm PVDF) and cooled to 15-25° C. Finally, an IPCassay was conducted.

Example 9

The solution prepared in Example 8 was lyophilized to provide areconstitutable and/or dilutable dry powder comprising melphalan as ahydrochloride salt. For the lyophilization, glass vials were filled withthe solution (10 mL) and placed in trays on a pre-cooled shelf at 5° C.The vials were allowed to thermally equilibrate for about 1 hour, andwere lyophilized to provide a dry powder in each vial. The vials wereback-filled with nitrogen, sealed, packaged, and labeled. The vials wereprotected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Prophetic Example B

The liquid pharmaceutical composition provided in Example 8 will beaseptically spray dried to provide a free-flowing powder to be filledaseptically. The free-flowing powder will meet or exceed the dissolutionproperties of the lyophilized powder prepared in Example 9.

Example 10

The properties of the pharmaceutical compositions of the presentdisclosure after dilution with Water for Injection, USP, were analyzedby a variety of analytical methods. The results are listed in the Tablebelow. Compositions A-D were prepared by the process described inExamples 8-9. The diluted compositions contained SBE_(6.5)-β-CD(CAPTISOL®) in a concentration of 75 mM, 100 mM, 125 mM, and 125 mM,respectively. Each of the compositions had a moisture content of about1.3% to about 2.5% prior to dilution.

TABLE Properties of pharmaceutical compositions of the presentdisclosure containing varying concentrations of a cyclodextrinderivative. Dilution SBE_(6.5)- Dissolution Density Time Volume β-CDConc. Time pH (22° C.) Viscosity for 5% loss A 10 mL  75 mM <30 s 5.051.07 g/cc 2.06 cP 10 h B 10 mL 100 mM <30 s 4.9 1.08 g/cc 2.28 cP 23 h C10 mL 125 mM  45 s 5.05 1.11 g/cc 2.95 cP 49 h D  5 mL 125 mM 105 s 5.21.11 g/cc 3.02 cP 25 h

Example 11

The stability of melphalan hydrochloride upon dilution of apharmaceutical composition of the present disclosure was determined.Pharmaceutical compositions containing a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.-2163 g/mol) were diluted withisotonic saline to provide 0.45 mg/mL melphalan solutions that containedthe cyclodextrin derivative at a concentration of 75 mM and 125 mM,respectively. Melphalan was assayed as a function of time, and the timenecessary for a 5% or 10% loss of melphalan (based on an initialmelphalan concentration of 100%) was determined. The data is provided inthe Table below. The times required for melphalan to fall to 90% or 95%of its initial concentration in the solutions that contained thecyclodextrin derivative were compared to the stability of melphalan in aReference product (ALKERAN® for Injection, GlaxoSmithKline).

TABLE Melphalan stability as a function of cyclodextrin derivativeconcentration, compared to a reference melphalan standard. Time for 5%loss Time for 10% loss SBE_(6.5)-β-CD (75 mM) 5.4 h 11 h SBE_(6.5)-β-CD(125 mM) 8.8 h 18 h Reference 1.3 h 2.7 h 

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentdisclosure shows an improvement of approximately 4.2 times and 6.8 timesat a cyclodextrin derivative concentration of 75 mM and 125 mM,respectively, compared to a reference melphalan formulation that doesnot contain a cyclodextrin derivative.

Example 12

The stability of melphalan hydrochloride upon dilution of apharmaceutical composition of the present disclosure was determined as afunction of temperature and storage conditions. Pharmaceuticalcompositions containing melphalan (50 mg) and a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg) were dilutedwith isotonic saline (8.5 mL) to provide a concentrated solution. Theconcentrated solution was further diluted 10-fold to provide a dilutesolution. Each of the concentrated and diluted melphalan solutions werestored at 25° C./60% relative humidity, or in a refrigerator (˜10° C.),and the melphalan content was monitored as a function of time. The datais provided in the Table below.

TABLE Melphalan stability as a function of temperature and storageconditions. Monohydroxy Storage Melphalan Melphalan Solution TimeConditions Assay Assay Cone. Sol'n 0 Refrigerator 99% 0.8% ″ 6.5 ″98.9%   0.8% ″ 24.5 ″ 98.8%   0.9% ″ 48.5 ″ 98.4%     1% Conc. Sol'n 025° C./60% R.H. 99% 0.8% ″ 6 ″ 98% 1.5% ″ 24 ″ 96% 3.4% ″ 48 ″ 93% 5.7%Dilute Sol'n 0 Refrigerator 99%   1% ″ 5 ″ 98.4%   1.4% ″ 24.3 ″ 97.8%    2% ″ 48.4 ″ 96.7%   2.7% Dilute Sol'n 0 25° C./60% R.H. 99%   1% ″ 5.3″ 94% 5.3% ″ 23.8 ″ 81% 15.5%  ″ 47.2 ″ 70% 20.2% 

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentdisclosure provides a significant improvement compared to currentlyavailable melphalan pharmaceutical compositions that do not contain acyclodextrin derivative.

Example 13

The stability of a lyophilized melphalan hydrochloride composition wasdetermined before and after dilution as a function of temperature andstorage conditions. The study was performed under the direction of CyDexPharmaceuticals, Inc. by BioConvergence LLC, Bloomington, Ind.

Compositions comprising melphalan (50 mg) and povidone (20 mg) werediluted with compositions comprising a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg), sodiumcitrate (200 mg), and distilled water (10 mL) to provide concentratedmelphalan solutions (5 mg/mL). In addition to testing the stability ofthe concentrated solutions, further dilution 11-fold to provide dilutesolutions containing melphalan (0.45 mg/mL).

The kinetic stability of a reconstituted concentrated solution (5 mg/mLmelphalan) was determined upon storage in a glass vial, and the kineticstability of a reconstituted diluted solution (0.45 mg/mL melphalan) wasdetermined upon storage in a 50 mL Baxter INTRAVIA® bag, at refrigerated(about 2°-8° C.) and room (about 25° C., monitored under fluorescentlight) temperatures.

Evaluation of the kinetic stability of the dilute (0.45 mg/ml melphalan)composition was determined in a 50 mL Type I glass container:reconstitution was performed using saline (8.5 mL), and aliquots (4.5mL) were removed from each vial and injected into 4 glass containersthat held 45.5 mL of saline. After an amount was withdrawn from eachcontainer for t=0 analysis, the containers were stored at roomtemperature (“RT”, about 25° C., under fluorescent light), or in arefrigerator (about 2°-8° C.), and the melphalan content was monitoredas a function of time. The data is provided in the Table below.

TABLE Summary of melphalan stability as a function of temperature andstorage conditions Hold MEL Degradation Run Form Storage Conditions Time(% w/w) 1 Lyophilized comp. RT (~20°-25° C.)  2 yrs <2% Conc. solution(5 mg/mL) Immediately diluted n/a n/a Dilute solution (0.45 RT (~20°-25°C.) 10 h <4% mg/mL) 2 Lyophilized comp. RT (~20°-25° C.)  2 yrs <2%Cone. solution (5 mg/mL RT (~20°-25° C.) 24 h <4% Dilute solution (0.45mg/mL) RT (~20°-25° C.)  5 h <2% 3 Lyophilized comp. RT (~20°-25° C.)  2yrs <2% Conc. solution (5 mg/mL) Refrigerated (2°-8° C.) 48 h <4% Dilutesolution (0.45 RT (~20°-25° C.)  5 h <2% mg/mL) 4 Lyophilized comp. RT(~20°-25° C.  2 yrs <2% Conc. solution (5 mg/mL) Immediately diluted n/an/a Dilute solution (0.45 1) Refrigerated (2°-8° C.) 1) 24 h 1) <2%mg/mL) 2) RT (~20°-25° C.) 2) 5 h 2) <2%

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentdisclosure provides a stable composition that can be maintained at roomtemperature for up to 5 hours and exhibit less than 2% melphalandegradation, or up to 10 hours and exhibit less than 4% melphalandegradation. When refrigerated, a diluted melphalan composition can bestored up to 24 hours and exhibit less than 2% melphalan degradation.Additionally, a lyophilized pharmaceutical composition can be stored upto 2 years at room temperature and exhibit less than 2% melphalandegradation.

Furthermore, referring to Examples 12 and 13, the pharmaceuticalcompositions of the present disclosure provide a significant improvementin melphalan stability compared to other formulations in which the useof a cyclodextrin derivative has been proposed. For example, D. Q. Ma etal., Int. J. Pharm. 189:227 (1999) provide a melphalan composition thatupon dilution with a solution containing a cyclodextrin derivative,exhibits a melphalan loss of more than 60% after 48 hours at roomtemperature. Significantly, the data in the above Tables illustratesthat upon dilution of a pharmaceutical composition of the presentdisclosure, a melphalan loss of at most 30% is observed within 48 hoursat room temperature. The melphalan loss can be reduced to as low as 2%or 3% when the solution is stored at a reduced temperature (e.g., in arefrigerator).

Example 14

The hemolytic potential of a cyclodextrin derivative suitable for usewith the pharmaceutical composition of the present disclosure wasanalyzed in comparison to a previously marketed diluent vehicle formelphalan (ALKERAN® For Injection, GlaxoSmithKline). The hemolyticpotential was evaluated in rodent (SPRAGUE DAWLEY® or Wistar Han IGSrats) and human red blood cells obtained from fasted subjects using aspectrophotometric technique. Normal saline (0.9% sodium chloride) wasused as the blank (or background) and as a negative control forcomparison against various concentrations of a cyclodextrinderivative-containing, and cyclodextrin-free diluent vehicles. Apositive control containing Triton X-100 (1%) in phosphate bufferedsaline was also utilized. Human red blood cells were taken from fasted(≥8 h) adult subjects. The components of the various samples are listedin the following Table.

TABLE Components of diluent vehicles used for hemolysis studies.Identification Constituents Negative Control/Blank 0.9% Sodium ChloridePositive Control 1% TRITON ® X-100 in phosphate buffered salineCyclodextrin Derivative SBE_(6.5)-β-CD (CAPTISOL ®, 9.72 g) Diluent q.s.400 mL with normal saline ALKERAN ® for Injection Povidone (K-12, 72 mg)(GlaxoSmithKline) Diluent Sodium citrate (720 mg) Propylene glycol (21.6mL, 22.4 g) Ethanol (1.87 mL, 1.48 g) Water (12.2 mL) q.s. 400 mL withnormal saline

The rat and human red blood cells were exposed to various concentrationsof the diluent vehicles and the hemolytic potential was evaluated usingequation (1):sA₅₄₀(test article)−A₅₄₀(negative control)×100=% Hemolysis  (1)

The hemolysis results are provided in the following Table, where Group Arefers to rat red blood cells exposed to the cyclodextrin derivativevehicle; Group B refers to rat red blood cells exposed to the ALKERAN®for Injection (GlaxoSmithKline) diluent vehicle; Group C refers to humanred blood cells exposed to the cyclodextrin derivative vehicle; andGroup D refers to human red blood cells exposed to the ALKERAN® forInjection (GlaxoSmithKline) diluent vehicle. The negative controls foreach experiment provided absorbances below 0.13, and the positivecontrols for each experiment provided absorbances of about 2.8 to 3.

TABLE Hemolysis results for rat and human red blood cells exposed tovarious diluent vehicles. Dilution Group Meas. none 1:2 1:4 1:8 1:161:32 1:64 1:128 A Abs. 0.11 0.114 0.11 0.117 0.116 0.118 0.119 0.118(a.u.) Hem. (%) 1% 0 0 0 0 0 0 0 B Abs. 0.178 0.155 0.141 0.128 0.1250.121 0.123 0.124 (a.u.) Hem. (%) 2% 1% 1% 0 0 0 0 0 C Abs. 0.021 0.0210.022 0.019 0.017 0.016 0.017 0.025 (a.u.) Hem. (%) 0 0 0 0 0 0 0 0 DAbs. 0.037 0.031 0.033 0.02 0.025 0.022 0.027 0.027 (a.u.) Hem. (%) 0 00 0 0 0 0 0

Referring to the hemolysis data in the above Table, the solution thatcontained the cyclodextrin derivative at high concentrations (e.g., nodilution, 1:2 dilution with saline, and 1:4 dilution with saline)provided reduced hemolysis in rat red blood cells, which was alsoexhibited as a reduction in spectrophotometric absorption of about 30%compared to the ALKERAN® for Injection (GlaxoSmithKline) diluent. Whilethe hemolysis tests in human blood cells exhibited a similar reductionin spectrophotometric absorbance at high concentrations, neither thecyclodextrin derivative solution or the ALKERAN® for Injection(GlaxoSmithKline) diluent vehicle induced hemolysis in human red bloodcells.

Example 15

A study was conducted that determined melphalan associated withSAE_(6.5)-β-CD (CAPTISOL®, CyDex Pharmaceuticals, Inc., Lenexa, Kans.)exhibits the same protein binding as unassociated melphalan. The studywas performed under the direction of CyDex Pharmaceuticals, Inc. byAnalytical Biochemistry Laboratories, Inc., Columbia, Mo.

Preliminary Study

A preliminary study was performed that determined radioactive-labeledmelphalan, [¹⁴C]-melphalan (Moravek Biochemicals, Inc., Brea, Calif.),does not bind non-specifically to ultrafiltration devices. The followingmixtures of compounds were added to human plasma ultrafiltrate(Biochemed, Winchester, Va.) to determine the protein binding of[¹⁴C]-melphalan alone or in combination with SAE_(6.5)-β-CD:

1. [¹⁴C]-melphalan with melphalan; and

2. [¹⁴C]-melphalan with melphalan and SAE_(6.5)-β-CD.

Radioactive-labeled warfarin, [³H]-warfarin (Moravek Biochemicals, Inc.,Brea, Calif.), a compound with well-documented protein bindingproperties was used as a positive control in all experiments.

Powdered material (as applicable) was weighed into scintillation vials(20 mL) and the radiolabeled compounds were added to the vials using apositive displacement pipette. Blank human plasma ultrafiltrate (5 mL)was then added to the vials using a glass serological pipette. Themixtures were then blended briefly. The time-dependence of plasmaprotein binding was determined by sampling the mixtures 0.5, 1, and 5minutes after addition of the test compounds to the human plasmaultrafiltrate. The sample aliquots (3×1 mL) were dispensed into theultrafiltration devices, and the samples were immediately centrifuged(1600 g for 5 minutes at 25° C.). The solution remaining in the vialswas then aliquoted for Liquid Scintillation Counting (LSC) analysis(2×0.1 mL).

More than 95% recovery was observed for [³H]-warfarin in allexperiments. In the protein-binding experiments, [³H]-warfarin was over99% protein bound. Radiolabeled melphalan (alone or in combination withSAE_(6.5)-β-CD) applied to ultrafiltration devices having a molecularweight cutoff of 30 kD exhibited an average of over 97% radioactivityrecovery: [¹⁴C]-melphalan alone exhibited a recovery of 97.7% (n=3), and[¹⁴C]-melphalan with SAE_(6.5)-β-CD exhibited a recovery of 97.6% (n=3).The results demonstrate that there was minimal (i.e., less than 2.4%)non-specific binding of radiolabeled melphalan to the ultrafiltrationdevices.

Protein-Binding Study

For the protein-binding study, the radiolabeled melphalan,non-radiolabeled melphalan, and SAE_(6.5)-β-CD (as applicable) wereadded to a scintillation vial (20 mL) and blown to dryness under anitrogen stream to standardize the amount of solvent present in eachexperiment. Methanol (50 μL) was added to the vials and blank humanplasma (5 mL) was added to the vials using a glass serological pipette.The mixtures were then blended briefly. Aliquots (3×1 mL) were thendispensed from the vials into the ultrafiltration devices, followed bycentrifugation (2,000 g for 5 minutes at 25° C.). The time intervalbetween adding human plasma to the vials and the start of centrifugationwas 0.5, 1, 5, 10, and 30 minutes. The solution remaining in the vialswas then aliquoted in duplicate (at a volume of 0.1, 0.05, and 0.025 mL)for LSC analysis.

Sample radioactivity was quantified using a scintillation counter(Beckman Instruments, Inc. Schaumberg, Ill.) equipped with the H-numbermethod for cpm to dpm conversion. LSC analysis was performed withsamples (5 mL) in glass scintiallation vials (7 mL), from whichbackground measurements were made using the same amount of scintillationfluid added to the vials. The results are provided in the followingTable:

TABLE Protein binding of radiolabeled melphalan (“[¹⁴C]-mel”) withunlabeled melphalan (“mel”) in the presence and absence ofSAE_(6.5)-β-CD. Total Recovery (%) % Binding Mixture Conc. (dpm/mL)Conc. (μg/mL) Rep. Data Avg. Data Avg. [¹⁴C]-mel + mel 112, 105 14 196.4 97.1 64.5 64.3 ± 0.34 (1 min.) 2 96.9 63.9 3 98.0 64.4 [¹⁴C]-mel +mel + 112, 382 14 1 92.9 91.5 63.7 64.3 ± 0.69 SAE_(6.5)-β-CD 2 95.464.1 (0.5 min) 3 86.4 65.1 [¹⁴C]-mel + mel + 108, 085 13 1 97.3 97.563.8 64.4 ± 0.59 SAE_(6.5)-β-CD 2 97.0 64.8 (1 min.) 3 98.3 64.6[¹⁴C]-mel + mel + 109, 621 13 1 96.5 95.9 65.8 67.2 ± 1.96SAE_(6.5)-β-CD 2 95.7 69.5 (5 min.) 3 96.5 66.4 [³H]-warfarin 114, 9320.052 1 98.5 101 98.9 98.8 ± 0.01 2 100 98.8 3 103 98.8

The results showed that [¹⁴C]-melphalan in the absence of SAE_(6.5)-β-CDwas 64.3% protein bound after 1 minute in human plasma. Similar degreesof protein binding were observed for [¹⁴C]-melphalan in the presence ofSAE_(6.5)-β-CD: 64.3% (0.5 minutes, n=3), 64.4% (1 minute, n=3), and67.2% (5 minutes, n=3). The study showed that SAE_(6.5)-β-CD did notaffect the protein-binding of radiolabeled [¹⁴C]-melphalan.

Example 16

A study was performed to investigate the potential for a sulfoalkylether cyclodextrin to perturb the in vivo pharmacokinetics of melphalan.Pharmacokinetic parameters were determined for melphalan followingintravenous administration to male Sprague Dawley rats in the presenceor absence of SBE_(6.5)-β-CD in the delivery vehicle.

The pharmacokinetics of melphalan were studied in overnight-fasted maleSprague Dawley rats. All experimental procedures were approved andperformed in accordance with the guidelines of the Institutional AnimalExperimentation Ethics Committee (Monash University Ethics approvalnumber VCPA/2008/02).

On the day prior to dosing, a commercially available BASi CULEX® cannula(for use with a CULEX® automated blood sampling device) was insertedinto the left carotid artery of each rat under isoflurane anesthesia(2%). A polyethylene cannula was also inserted into the right jugularvein. Cannulae were exteriorized by tunneling subcutaneously to emergeabove the scapulae.

Immediately following surgery and through to the end of the experiment,rats were housed in RATURN® metabolic cages in the CULEX® automatedblood sampler. All rats returned to normal grooming, drinking andsleeping behavior within an hour of surgery. Animals were given a smallamount of food just after they awoke from the anesthetic, but were thenfasted for 16-18 hours prior to drug administration. Animals had accessto water ad libitum at all times. Food was reinstated 4 hours followingdrug administration. At the conclusion of each experiment, rats werekilled by a single lethal injection of pentobarbitone.

The cyclodextrin derivative-free melphalan formulation was prepared asper the product insert for ALKERAN® for Injection (GlaxoSmithKline). Thecontents of a single ALKERAN® for Injection (GlaxoSmithKline) vial werereconstituted with 10 mL of sterile diluent (provided with the ALKERAN®product, and containing 0.2 g sodium citrate, 6 mL propylene glycol,0.52 mL ethanol (96%) and water). The solution was then further dilutedwith 0.9% normal saline (2 mL ALKERAN® for Injection (GlaxoSmithKline)in 10 mL of 0.9% saline, i.e., 12 mL total volume), and the resultantformulation was sterilized by filtering through a 0.22 μm syringe filterbefore administering to rats. The measured concentration of melphalan inthe IV formulation was 0.54 mg/mL (as free base) and the pH of the finalsolution was between 5 and 6 (checked using pH paper). The formulationwas administered to animals within 30 minutes of preparation.

A formulation containing SBE_(6.5)-β-CD (27% w/v) was prepared bydissolving SBE_(6.5)-β-CD in Milli-Q water. Contents of an ALKERAN® forInjection (GlaxoSmithKline) vial were then reconstituted with 10 mL ofthe SBE_(6.5)-β-CD solution. This solution was then diluted with 0.9%saline (2 mL in 10 mL of 0.9% saline) and the resultant formulation wassterilized by filtering through a 0.22 μm syringe filter beforeadministering to rats. Thus, the final formulation contained 4.5% (w/v)SBE_(6.5)-β-CD. The measured concentration of melphalan (as free base)was 0.58 mg/mL and the pH of the final solution was between 5 and 6(checked using pH paper). The formulation was administered to animalswithin 30 minutes of preparation.

The total dose volume was 1 mL and all doses were infused manually viathe jugular vein cannula. The complete dose was delivered over a10-minute period and the cannula was flushed with heparinized saline (10U/mL) to ensure complete administration of the dose.

Samples of arterial blood and urine were collected according to thefollowing schedules: blood/plasma sampling times were pre-dose, and 5,10 (end of infusion), 15, 25, 40, 55, 70, 100, 130, 190, 250, 370, and490 minutes post-dose; and urine sampling intervals were 0-70 minutes,70-130 minutes, 130-190 minutes, 190-250 minutes, 250-310 minutes,310-370 minutes, 370-430 minutes, 430-490 minutes, and 490-1450 minutes.

Arterial blood was collected directly into borosilicate vials (at 4° C.)containing heparin, COMPLETE® (a protease inhibitor cocktail), potassiumfluoride, and EDTA to minimize potential for ex vivo degradation inblood/plasma samples. Once collected, an aliquot (50 μL) of whole bloodwas transferred into a fresh micro-centrifuge tube. The remaining bloodwas centrifuged and supernatant plasma removed. All blood, plasma andurine samples were immediately (snap) frozen on dry ice and thentransferred to −20° C. freezer for storage until analysis.

The melphalan concentration in whole blood, plasma, urine and samples ofdosing solutions were determined using LC-MS.

Sample preparation was performed using protein precipitation withacetonitrile. Aliquots of the plasma and blood (50 μL) were treated withinternal standard (10 μL), acetonitrile (130 μL), vortexed andcentrifuged. The supernatant was removed and analyzed by LC/MS. Standardsamples were prepared by spiking solution standards in the respectiveblank matrix. A stock solution of melphalan free base was prepared at aconcentration of 10 mg/mL in dimethylsulfoxide. This stock solution wasfurther diluted in aqueous acetonitrile (50% v/v) to obtain spikingsolutions for the preparation of calibration standards.

All samples were assayed via LC-MS/MS on a Micromass Quattro Premier PRtriple quadrupole instrument coupled with a Waters Acquity UPLC (WatersCorp., Milford, Mass.). Analytical separations were performed on aPhenomenex Polar reverse-phase column (50 mm×1.0 mm inner diameter, 4 μmparticle size) equipped with a Phenomenex Polar Security Guard column ofthe same material (Torrance, Calif.). Samples (7.5 μL) were injectedonto the column and compounds were eluted (at a flow rate of 0.15mL/min) using a ternary gradient solvent system consisting of an aqueoussolution of methanol (2% v/v) and formic acid (0.05% v/v) in Milli-Qwater (solvent A), and acetonitrile containing formic acid (0.05% v/v)(solvent B). The gradient conditions used for LC-MS analysis are listedin the following table.

TABLE Gradient chromatography conditions used for the analysis ofmelphalan. Time (minutes) Solvent A Solvent B   0-0.2 89 2 0.3 80 20 2.720 80 2.8 5 95 3.3 5 95 3.5-4.5 98 2

Elution of analytes was confirmed by multiple reaction monitoring (MRM)using diazepam (0.2 μg/mL) as the internal standard (diazepam). Inletcone voltages were 20 eV and 40 eV for melphalan and the internalstandard, respectively, and collision energies of 15 eV and 27 eV formelphalan and the internal standard, respectively. The elution ofmelphalan and internal standard was monitored using the followingtransitions 304.94>267.88 and 285.17>154.02, respectively. Melphalanexhibited a retention time of 2.0 minutes and the internal standardexhibited a retention time of 2.8 minutes.

Mass spectrometry was performed using positive mode electrosprayionization with a capillary voltage of 3.2 kV, detector multiplier gainof 650 V, and source block and desolvation temperatures of 90° C. and300° C., respectively. A desolvation gas (nitrogen) and collision gas(argon) flow of 500 L/h and 0.38 mL/min, respectively, was maintained.The lower limit of quantitation (LLQ) for blood and plasma standards was5.0 ng/mL, and the LLQ for diluted urine samples was 0.5 ng/m L.

Both plasma and blood concentration data were analysed to obtainpharmacokinetic parameters using WINNONLIN® software (WINNONLIN®professional version 5.2.1, Pharsight Corp., Mountain View, Calif.). Thetotal clearance (CL_(total), for whole blood or plasma) afterintravenous administration was calculated as: CL_(total)=Dose/AUC, whereAUC is the area under the whole blood or plasma concentration versustime curve obtained using the linear trapezoidal method. The volume ofdistribution (V_(Z)) was calculated as: V_(Z)=CL_(total)/λ_(Z), whereλ_(Z), is the elimination rate constant after i.v. administration.

The mean dose-normalized concentration versus time profiles of melphalanin whole blood and plasma following intravenous administration using aformulation containing SBE_(6.5)-β-CD (n=4) and a cyclodextrin-free(n=5) formulation are presented in the following table.

TABLE Pharmacokinetic parameters for melphalan in whole blood and plasmafollowing intravenous administration to male Sprague Dawley rats at anominal dose of 2.0 mg/kg with a formulation containing a cyclodextrinderivative (SBE_(6.5)-β-CD) and a formulation free from a cyclodextrinderivative (“CD-free formulation”). Whole Blood Plasma SBE_(6.5)-β-SBE_(6.5)-β- CD CD-free CD CD-free Melphalan (27% w/v) formulation (27%w/v) formulation Apparent t_(1/2) (h) 0.8 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.9± 0.1 CL_(total) 10.9 ± 2.0  12.2 ± 1.3  8.1 ± 1.1 9.3 ± 1.4 (mL/min/kg)V_(z) (L/kg) 0.8 ± 0.2 1.0 ± 0.2 0.6 ± 0.1 0.7 ± 0.2 AUC_(0-inf)/D 94.2± 16.6 82.8 ± 8.8  125.4 ± 15.0  109.6 ± 16.1  (μM · min · kg/μmol)

FIGS. 5A-5B provide graphic representations of the dose-normalized wholeblood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalanfollowing intravenous administration for the SBE_(6.5)-β-CD-containingmelphalan formulation (●), n=4, and the cyclodextrin-free (∘), n=5,melphalan formulation (ALKERAN® for Injection, GlaxoSmithKline).Referring to FIGS. 5A-5B, the data is presented as mean with error barsindicating a single standard deviation. Melphalan exhibitedbi-exponential pharmacokinetics in both whole blood and plasma and theapparent terminal elimination phase was well-defined within the 8 hourpost-dose blood sampling period, with the apparent terminal eliminationhalf-life. The mean whole blood and plasma concentration versus timeprofiles for the formulation that contained SBE_(6.5)-β-CD (27% w/v) andcyclodextrin-free formulation were essentially super-imposable and therewere no statistically significant differences in any of thepharmacokinetic parameters between the two formulations (p>0.05). Thus,as shown in the above table, in the rat the in vivo pharmacokineticparameters for a melphalan formulation containing SBE_(6.5)-β-CD areessentially identical to the pharmacokinetic parameters for acyclodextrin-free formulation (i.e., ALKERAN® for Injection,GlaxoSmithKline).

Additionally, in both formulations the percentage of the melphalan doseexcreted in urine excreted as unchanged compound up to 24 hourspost-dose was low: for the cyclodextrin-free formulation the average was2.7±1.7%, and for the formulation containing SBE_(6.5)-β-CD the averagewas 2.3±2%.

The data show that pharmacokinetic parameters, including half-life, AUC,volume of distribution, clearance, and the extent of renal eliminationof melphalan were essentially unchanged between ALKERAN® for Injection(GlaxoSmithKline) and the melphalan formulation that containedSAE_(6.5)-β-CD. Specifically, the mean whole blood and plasmaconcentration versus time profile of melphalan with and withoutSBE_(6.5)-β-CD are essentially super-imposable. The results demonstratethat SBE_(6.5)-β-CD had no observable difference in blood or plasmaversus time profiles for melphalan in the rat model. Furthermore, therewas no apparent difference in the urinary excretion of melphalan in therat model.

Example 17

A Phase IIa, multicenter, open-label, randomized, efficacy and safetystudy of melphalan hydrochloride administered by injection using apropylene glycol-free diluent vehicle has been conducted in 3 humanmultiple myeloma patients who underwent myeloablative conditioning inpreparation for autologous transplantation. The study is on-going.

The primary goal of the study was/is to determine the rate ofmyeloablation and neutrophil engraftment in multiple myeloma patientswho receive a high-dose of melphalan hydrochloride via injection inwhich one dose is administered using a propylene glycol diluent and onedose is administered using a propylene glycol-free diluent. Theadministering is used as myeloablative therapy prior to autologous stemcell transplantation.

The secondary goal of the study is to determine: (a) the rate ofplatelet engraftment in multiple myeloma patients receiving high-dosemelphalan hydrochloride via injection using both a propylene glycoldiluent and a propylene glycol-free diluent prior to autologous stemcell transplantation; (b) the median time to engraftment of neutrophiland platelets in multiple myeloma patients receiving high-dose melphalanhydrochloride via injection using both a propylene glycol diluent and apropylene glycol free diluent prior to autologous stem celltransplantation; (c) the response rate (stringent complete response[sCR], complete response [CR], very good partial response [VGPR],partial response [PR], stable disease [SD], or progressive disease [PD])at autologous stem cell transplantation at day +100 in multiple myelomapatients receiving high-dose melphalan hydrochloride via injection usinga propylene glycol diluent and a propylene glycol-free diluent prior toautologous stem cell transplantation; (d) the toxicity profile ofhigh-dose melphalan hydrochloride via injection using both a propyleneglycol diluent and a propylene glycol-free diluent in multiple myelomapatients undergoing autologous stem cell transplantation; (e) the rateof treatment-related mortality during the first 100 days afterautologous stem cell transplantation in multiple myeloma patientsreceiving high-dose melphalan hydrochloride via injection using apropylene glycol diluent and a propylene glycol-free diluent; and (f)the pharmacokinetics of melphalan hydrochloride via injection using apropylene glycol diluent compared with the pharmacokinetics of melphalanhydrochloride via injection using a propylene glycol-free diluent (i.e.,a cyclodextrin derivative) in multiple myeloma patients undergoingautologous stem cell transplantation.

Patients were screened prior to enrollment in the study. Patients fromany of the following classes were qualified for inclusion in the study:

Patients with symptomatic multiple myeloma requiring treatment atdiagnosis or anytime thereafter;

Patients with multiple myeloma who qualify for autologous stem celltransplantation therapy who have received appropriate primary inductiontherapy for transplantation;

Patients who are 70 years of age or younger at time of transplantation(patients greater than 70 years of age may qualify on a case-by-casebasis if the patient meets criterion based on institution's standard ofpractice);

Patients with an adequate autologous graft, defined as anun-manipulated, cryopreserved, peripheral blood stem cell or bone marrowstem cell graft containing at least 2×10⁶ CD34+ cells/kg based uponpatient weight, along with a reserve of 2×10⁶ CD34+ cells/kg that isstored in a separate bag; and

Patients with adequate organ function as measured by:

-   -   Cardiac: Left ventricular ejection fraction at rest >40%;    -   Hepatic: Bilirubin <2× the upper limit of normal and        ALT/AST<3×ULN;    -   Renal: Creatinine clearance >40 mL/minute; and    -   Pulmonary: DLCO, FEV₁, FVC>50% of predicted value (corrected for        Hgb) or O₂ saturation >92% on room air.

All patients have received antiemetics, hydration, and infectionprophylaxis according to institutional guidelines. Patients followedinstitutional guidelines regarding hospitalization. Patients returnedfor daily laboratory tests (CBC with differential and platelets and abasic chemistry panel) until neutrophil engraftment, and then returnedfor weekly safety evaluations until autologous stem cell transplantationDay +30. The following safety, efficacy, and pharmacokinetic evaluationswere performed prior to the first dose of melphalan, and at thefollowing post-dose time points:

-   -   Twelve blood samples were taken at specific time points for a        pharmacokinetic evaluation. Blood samples were collected        immediately prior to and after receiving the melphalan dose;    -   Vital signs were recorded hourly during the first eight hours        after receiving each dose of melphalan, then repeated once daily        until hospital discharge, and then weekly until Day +30. Weight        were collected at hospital discharge and at Day +30;    -   A 12-lead electrocardiograph, (ECG) along with a 10 to 20 second        rhythm strip was collected twice weekly until hospital        discharge, then a 12-lead ECG (without a rhythm strip) was        collected weekly until Day +30;    -   A focused physical examination was performed daily until        hospital discharge, then a complete physical examination was        performed weekly until Day +30;    -   Toxicity grading and evaluation for AEs/SAEs was according to        NCI-CTC AE Version 3.0 during the entire Study Period;    -   Complete blood count with differential and platelet count was        performed daily until neutrophil and platelet engraftment, then        weekly until Day +30;    -   Eastern Cooperative Group performance status was examined at the        time of hospital discharge, then weekly until Day +30;    -   Basic serum chemistry panel (sodium, potassium, chloride,        glucose, creatinine, bicarbonate, and BUN) daily until        neutrophil engraftment;    -   Full serum chemistry panel (sodium, potassium, chloride,        magnesium, bicarbonate, glucose, total protein, albumin,        calcium, phosphate, uric acid, BUN, creatinine, CPK, total        bilirubin, alkaline phosphatase, LDH, SGOT, and SGPT) will be        monitored weekly until Day +30;    -   Urinalysis (specific gravity, pH, protein, glucose, ketones,        nitrite, RBCs, and WBCs) was monitored twice weekly until        hospital discharge, then weekly until Day +30; and    -   Concomitant medications was recorded during the entire study        period.

A melphalan dose of 200 g/m² was divided into two separate, consecutivedoses of 100 mg/m² administered on two separate days (Day −3 and Day −2)prior to the patients receiving an autologous stem cell transplantation.For the calculation of body surface area, actual body weight was usedfor patients who weighed less than or between 100% to 130% of theirideal body weight. Patients who weighed more than 130% of their idealbody weight were dosed based on a body surface area obtained bycalculating the patient's adjusted body weight.

Patients were randomly chosen to receive the first melphalan dose of 100mg/m² (on Day −3) via either a composition comprising a propylene glycoldiluent (i.e., Melphalan HCl Injectable, Bioniche Pharma USA) or acomposition comprising a cyclodextrin derivative (SBE_(6.5)-β-CD,CAPTISOLe, at a concentration of 125 mM). Patients who randomly receivedthe first melphalan dose of 100 mg/m² as a composition comprising acyclodextrin derivative (on Day −3) then received a second melphalandose of 100 mg/m² (on Day −2) using the composition comprising apropylene glycol diluent. Conversely, patients who randomly received thefirst melphalan dose of 100 mg/m² as a composition comprising apropylene glycol diluent (on Day −3) then received a second melphalandose of 100 mg/m² (on Day −2) using the composition comprising acyclodextrin derivative.

For the composition comprising a cyclodextrin derivative, a dry powdercomposition containing melphalan as a hydrochloride salt was dilutedwith normal saline to a melphalan concentration no greater than 0.45mg/mL and a cyclodextrin concentration of 125 mM. The diluted solutionwas infused over 60 minutes via a central venous catheter.

The composition comprising a propylene glycol diluent was administeredusing a cyclodextrin-free composition (Melphalan HC1 Injectable,Bioniche Pharma USA) using the protocol described herein supra.

Following one day of rest after the myeloablative conditioning (Day −1),patients received an autologous graft with a minimum cell dose of 2×10⁶CD34+ cells/kg of patient body weight (Day 0). Cryopreservation andthawing of the product was consistent with Foundation for theAccreditation of Cellular Therapy standards and local institutionalpractice. The graft was infused per institutional protocol. Starting onDay +5, G-CSF was be administered at a dose of 5 μg/kg/day untilabsolute neutrophil count was greater than 500/mm³.

Blood samples for pharmacokinetic evaluation of melphalan were collectedafter each dose of melphalan and the pharmacokinctic parameters for invivo melphalan distribution were evaluated. Samples for evaluation ofthe pharmacokinetic parameters were collected by taking 5 mL venousblood samples immediately prior to melphalan administration and at 0,10, 20, 30, 60, 90, 120, 180, 240, 360, and 480 minutes following theend of the melphalan infusion. Pharmacokinetic parameters weredetermined by nonparametric pharmacokinetic data analysis techniques.Pharmacokinetic parameters computed from plasma drug concentration-timedata include the following:

-   -   C_(max), derived from the individual raw data;    -   T_(max), derived from the individual raw data;    -   Apparent terminal first-order elimination rate constant        (k_(el));    -   Apparent elimination t_(1/2);    -   Area under the plasma concentration-time curve to the last        measurable time point (AUC_(0-t)), calculated by the trapezoidal        rule; and    -   Area under the plasma concentration-time curve from the last        measurable time point extrapolated to infinity (AUC_(t-∞)),        determined from the concentration at the last measurable time        point divided by the k_(el).

The plasma concentrations and pharmacokinetic parameters were summarizedusing descriptive statistics. The data from patients 1-3 is shown in thefollowing table.

TABLE Individual patient melphalan pharmacokinetic parameters afterintravenous administration of a melphalan formulation that containedSBE6.5-β-CD and a cyclodextrin-free melphalan formulation (i.e.,Melphalan HC1 Injectable, Bioniche Pharma USA). SBE_(6.5)-β-CD CD-freePatient Parameter (27% w/v) formulation Ratio 1^(a) C_(max) (ng/mL)3,230 2,160 1.50 T_(max) (min) 10 20 — AUC_(0-t) 259,073 202,714 1.28AUC_(0-∞) 264,656 208,028 1.27 λ_(z)(min⁻¹) 0.0105 0.0103 — t_(1/2)(min)65.8 67.1 — 2^(b) C_(max) (ng/mL) 2,730 2,010 1.36 T_(max) (min) 10 10 —AUC_(0-t) 198,051 151,456 1.31 AUC_(0-∞) 202,728 154,130 1.32λ_(z)(min⁻¹) 0.0103 0.0113 — t_(1/2)(min) 67.4 61.6 — 3^(a) C_(max)(ng/mL) 4,590 2,890 1.59 T_(max) (min) 10 10 — AUC_(0-t) 306,432 230,6811.33 AUC_(0-∞) 314,108 236,059 1.33 λ_(z)(min⁻¹) 0.0101 0.0104 —t_(1/2)(min) 68.5 66.4 — ^(a)Patients 1 and 3 were administered theSBE_(6.5)-β-CD-containing formulation on Day −3 and the CD-freeformulation on Day −2. ^(b)Patient 2 was administered the CD-freeformulation on Day −3 and the SBE_(6.5)-β-CD-containing formulation onDay −2.

FIG. 6, provides a graphic representation of the mean plasma melphalanconcentration in a human patient after intravenous administration of amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and after intravenous administration of acyclodextrin-free melphalan formulation (Melphalan HCl Injectable,Bioniche Pharma USA). Referring to FIG. 6 and the data in the abovetable, the in vivo distribution of melphalan administered with asulfoalkyl ether cyclodextrin derivative provides a nearly 50% increasein the maximum in vivo concentration of melphalan, and approximately a30% increase in the area under the plasma concentration curves (i.e.,for both AUC_(0-t) and AUC_(0-∞)). As shown in the table above, the datafor patients 2 and 3 exhibited similar pharmacokinetic results. In viewof pharmacokinetic data obtained for these melphalan formulations in therat model, the enhancement in C_(max) and AUC for theSBE_(6.5)-β-CD-containing melphalan formulation in human patients iswholly unexpected.

As noted above, the study is on-going. The primary efficacy end points,which will be based on an intent-to-treat analysis of all patients, willbe the rate of myeloablation and the rate neutrophil engraftment. Thefollowing definitions will be used for these end points:

Myeloablation will be defined as any of the following:

-   -   Absolute neutrophil count less than 0.5×10⁹/L;    -   Absolute lymphocyte count less than 0.1×10⁹/L; or    -   Platelet count less than 20,000/mm³ or bleeding requiring        transfusion.

The first of two consecutive days for which cell counts drop below thesecut-off levels will be recorded as the date of myeloablation.

Neutrophil engraftment is defined as absolute neutrophil count greaterthan 0.5×10⁹/L on three consecutive daily assessments.

Secondary efficacy end points will be based on the following criteria:

-   -   The rate of platelet engraftment, which will be defined as an        un-transfused platelet measurement >20,000/mm³ on three        consecutive daily assessments;    -   The time to neutrophil engraftment, which will be defined as the        first of three assessments where absolute neutrophil count is        greater than 0.5×10⁹/L;    -   The time to platelet engraftment, which will be defined as the        first of three consecutive daily assessments where un-transfused        platelet measurement is greater than 20,000/mm³;    -   The rate of non-engraftment, which will be defined as a failure        to reach an absolute neutrophil count greater than 0.5×10⁹/L on        three consecutive daily assessments by autologous stem cell        transplantation Day +100;    -   The rate of late graft failure or late rejection, which will be        defined as development of absolute neutrophil count less than        0.5×10⁹/L after having engrafted within the first 100 days;    -   The rate of multiple myeloma response (sCR, CR, VGPR, PR, SD, or        PD), which will be defined according to International Working        Group criteria at Day +100; and    -   The rate of treatment-related mortality, which will be defined        as death without relapse or progression at Day +100.

The clinical trial is expected to demonstrate that melphalanadministered with a cyclodextrin derivative (SBE_(6.5)-β-CD) istherapeutically effective and safe for use in subjects for whom a stemcell transplantation has been indicated as conditioning prior to stemcell transplantation.

Example 18

A Phase IIb, multi-center, open-label, non-randomized, efficacy andsafety study of melphalan hydrochloride administered by injection usinga propylene glycol-free vehicle will be conducted in human multiplemyeloma patients who have symptomatic multiple myeloma and qualify forASCT.

The parameters of the study will be similar to those described inExample 17, except that all patients will be administered a propyleneglycol-free melphalan composition (100 mg/m²) on Day −3 and Day −2 usinga melphalan composition that includes a cyclodextrin derivative.Otherwise, the inclusion criteria, exclusion criteria, safety criteria,dosing, treatment, and efficacy endpoints will be similar to thosedescribed above in Example 17.

Example 19

A pharmaceutical composition comprising mechlorethamine as ahydrochloride salt is prepared by the process described in Example 4,except that mechlorethamine is used in place of melphalan.

Example 20

The liquid pharmaceutical composition provided in the previous exampleis lyophilized to provide a reconstitutable and/or dilutable dry powdercomprising about 50 mg of mechlorethamine as a hydrochloride salt. Glassvials are filled with the solution (10 mL) and placed in trays on apre-cooled shelf at 5° C. The vials are allowed to thermally equilibratefor about 30 minutes, and are then lyophilized to provide a dry powderin each vial. The vials are back-filled with nitrogen at a pressure ofabout 400 mTorr, and then sealed.

Example 21

A pharmaceutical composition comprising mechlorethamine as ahydrochloride salt is prepared by the process described in Example 8,except that mechlorethamine is used in place of melphalan.

Example 22

The solution prepared in the previous example is lyophilized to providea reconstitutable and/or dilutable dry powder comprising mechlorethamineas a hydrochloride salt. For the lyophilization, glass vials are filledwith the solution (10 mL) and placed in trays on a pre-cooled shelf at5° C. The vials are allowed to thermally equilibrate for about 1 hour,and are lyophilized to provide a dry powder in each vial. The vials areback-filled with nitrogen, sealed, packaged, and labeled. The vials areprotected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Example 23

A pharmaceutical composition comprising cyclophosphamide as ahydrochloride salt is prepared by the process described in Example 4,except that cyclophosphamide is used in place of melphalan.

Example 24

The liquid pharmaceutical composition provided in the previous exampleis lyophilized to provide a reconstitutable and/or dilutable dry powdercomprising about 50 mg of cyclophosphamide as a hydrochloride salt.Glass vials are filled with the solution (10 mL) and placed in trays ona pre-cooled shelf at 5° C. The vials are allowed to thermallyequilibrate for about 30 minutes, and are then lyophilized to provide adry powder in each vial. The vials are back-filled with nitrogen at apressure of about 400 mTorr, and then sealed.

Example 25

A pharmaceutical composition comprising cyclophosphamide as ahydrochloride salt is prepared by the process described in Example 8,except that cyclophosphamide is used in place of melphalan.

Example 26

The solution prepared in Example the previous example is lyophilized toprovide a reconstitutable and/or dilutable dry powder comprisingcyclophosphamide as a hydrochloride salt. For the lyophilization, glassvials are filled with the solution (10 mL) and placed in trays on apre-cooled shelf at 5° C. The vials are allowed to thermally equilibratefor about 1 hour, and are lyophilized to provide a dry powder in eachvial. The vials are back-filled with nitrogen, sealed, packaged, andlabeled. The vials are protected from exposure to light during allaspects of the lyophilization, back-filling, sealing, packaging andlabeling procedures.

Example 27

A pharmaceutical composition comprising ifosfamide as a hydrochloridesalt is prepared by the process described in Example 4, except thatifosfamide is used in place of melphalan.

Example 28

The liquid pharmaceutical composition provided in the previous exampleis lyophilized to provide a reconstitutable and/or dilutable dry powdercomprising about 50 mg of ifosfamide as a hydrochloride salt. Glassvials are filled with the solution (10 mL) and placed in trays on apre-cooled shelf at 5° C. The vials are allowed to thermally equilibratefor about 30 minutes, and are then lyophilized to provide a dry powderin each vial. The vials are back-filled with nitrogen at a pressure ofabout 400 mTorr, and then sealed.

Example 29

A pharmaceutical composition comprising ifosfamide as a hydrochloridesalt is prepared by the process described in Example 8, except thatifosfamide is used in place of melphalan.

Example 30

The solution prepared in Example the previous example is lyophilized toprovide a reconstitutable and/or dilutable dry powder comprisingifosfamide as a hydrochloride salt. For the lyophilization, glass vialsare filled with the solution (10 mL) and placed in trays on a pre-cooledshelf at 5° C. The vials are allowed to thermally equilibrate for about1 hour, and are lyophilized to provide a dry powder in each vial. Thevials are back-filled with nitrogen, sealed, packaged, and labeled. Thevials are protected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Example 31

A pharmaceutical composition comprising bendamustine as a hydrochloridesalt is prepared by the process described in Example 4, except thatbendamustine is used in place of melphalan.

Example 32

The liquid pharmaceutical composition provided in the previous exampleis lyophilized to provide a reconstitutable and/or dilutable dry powdercomprising about 50 mg of bendamustine as a hydrochloride salt. Glassvials are filled with the solution (10 mL) and placed in trays on apre-cooled shelf at 5° C. The vials are allowed to thermally equilibratefor about 30 minutes, and are then lyophilized to provide a dry powderin each vial. The vials are back-filled with nitrogen at a pressure ofabout 400 mTorr, and then sealed.

Example 33

A pharmaceutical composition comprising bendamustine as a hydrochloridesalt is prepared by the process described in Example 8, except thatbendamustine is used in place of melphalan.

Example 34

The solution prepared in Example the previous example is lyophilized toprovide a reconstitutable and/or dilutable dry powder comprisingbendamustine as a hydrochloride salt. For the lyophilization, glassvials are filled with the solution (10 mL) and placed in trays on apre-cooled shelf at 5° C. The vials are allowed to thermally equilibratefor about 1 hour, and are lyophilized to provide a dry powder in eachvial. The vials are back-filled with nitrogen, sealed, packaged, andlabeled. The vials are protected from exposure to light during allaspects of the lyophilization, back-filling, sealing, packaging andlabeling procedures.

CONCLUSION

These examples illustrate possible embodiments of the present invention.While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. It will be apparent to persons skilledin the relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

What is claimed is:
 1. An injectable aqueous pharmaceutical formulationcomprising 8.5 mL of an aqueous solution at a pH of 5, said solutionconsisting of 9 g/L sodium chloride, a sulfobutyl ether-β-cyclodextrin,and 5 mg/mL melphalan, wherein said sulfobutyl ether-β-cyclodextrin andsaid melphalan are present in a weight ratio of 54:1; and wherein saidaqueous pharmaceutical formulation is stable at room temperature for atleast one hour, as compared to a cyclodextrin-free reference melphalanstandard.
 2. The aqueous formulation of claim 1, wherein less than 2% byweight of said melphalan is degraded after 5 hours at room temperatureas compared to the amount of melphalan present before holding saidformulation at room temperature for 5 hours.
 3. The aqueous formulationof claim 1, wherein less than 4% by weight of said melphalan is degradedwhen said formulation is held at room temperature for 10 hours ascompared to the amount of melphalan present before holding saidformulation at room temperature for 10 hours.
 4. An injectable aqueouspharmaceutical formulation comprising 10 mL of an aqueous solution witha pH of 5, said formulation consisting of a sulfobutylether-β-cyclodextrin and 0.45 mg/mL melphalan; wherein said sulfobutylether-β-cyclodextrin and said melphalan are present in a weight ratio ofat least 54:1; and wherein said aqueous pharmaceutical formulation isstable at room temperature for at least 4 hours, as compared to acyclodextrin-free reference melphalan standard.
 5. The aqueousformulation of claim 4, wherein less than 2% by weight of said melphalanis degraded when said formulation is held at room temperature for 5hours as compared to the amount of melphalan present before holding saidformulation at room temperature for 5 hours.
 6. The aqueous formulationof claim 4, wherein less than 4% by weight of said melphalan is degradedwhen said formulation is held at room temperature for 10 hours ascompared to the amount of melphalan present before holding saidformulation at room temperature for 10 hours.
 7. An injectable aqueouspharmaceutical formulation comprising 8.5 mL of an aqueous solution at apH of 5, said solution consisting of at least one of an antioxidant,preservative, or buffer, 9 g/L sodium chloride, a sulfobutylether-β-cyclodextrin, and 5 mg/mL melphalan, wherein said sulfobutylether-β-cyclodextrin and said melphalan are present in a weight ratio of54:1; wherein said aqueous pharmaceutical formulation is stable at roomtemperature for at least one hour, as compared to a cyclodextrin-freereference melphalan standard.
 8. The aqueous formulation of claim 1 or 4or 7, wherein administration of the formulation provides up to a 30%increase in the area under the melphalan plasma concentration curve ascompared to administration of a cyclodextrin-free melphalan formulation.9. An injectable aqueous pharmaceutical formulation consisting of 5mg/mL melphalan, sulfobutyl ether-β-cyclodextrin and 9 g/L sodiumchloride; wherein said sulfobutyl ether-β-cyclodextrin and saidmelphalan are present in a weight ratio of 54:1; wherein the aqueouspharmaceutical formulation has a pH of 5; wherein said aqueouspharmaceutical formulation is stable at room temperature for at leastone hour, as compared to a cyclodextrin-free reference melphalanstandard; and further wherein administering the formulation provides amelphalan AUC_(0-t) in a subject that is at least 20% greater than amelphalan AUC_(0-t) provided by a melphalan formulation containing anequivalent dose of melphalan and lacking the cyclodextrin derivative.10. An injectable aqueous pharmaceutical formulation consisting of 0.45mg/mL melphalan, sulfobutyl ether-β-cyclodextrin and 9 g/L sodiumchloride; wherein said sulfobutyl ether-β-cyclodextrin and saidmelphalan are present in a weight ratio of at least 54:1; wherein theaqueous pharmaceutical formulation has a pH of 5; wherein said aqueouspharmaceutical formulation is stable at room temperature for at least 4hours, as compared to a cyclodextrin-free reference melphalan standard;and further wherein administering the formulation provides a melphalanAUC_(0-t) in a subject that is at least 20% greater than a melphalanAUC_(0-t) provided by a melphalan formulation containing an equivalentdose of melphalan and lacking the cyclodextrin derivative.